Improved methods of cell culture for adoptive cell therapy

Abstract

To further improve the efficiency and practicality of cell production.SOLUTION: Provided is an improved cell culture method for cell therapy applications, the method comprising growing cells of interest under the presence of antigen presenting cells and / or feeder cells. If the growth surface is not composed of a gas permeable material, the ratio of the culture medium volume to the surface area up to 1 ml / cm2 is used, and if the growth surface is composed of gas permeable material, the ratio of the culture medium volume to the surface area up to 2 ml / cm2 is used. The target cell has a surface density of 0.5×106 / cm2 or less at the start of the production cycle, and the sum of the surface density of the target cells and the surface density of the antigen presenting cells and / or the feeder cells is at least about 1.25×105 / cm2.SELECTED DRAWING: Figure 22D

Description

[Technical Field]

[0001] Related patent applications This application is a continuation-in-part of U.S. Patent Application No. 12 / 963,597, entitled "IMPROVED METHODS OF CELL CULURE FOR ADOPTIVE CELL THERAPY," filed December 8, 2010 (hereinafter the "Parent Application"), which claims priority to U.S. Provisional Patent Application No. 61 / 267,761, entitled "IMPROVED METHODS OF CELL CULURE FOR ADOPTIVE CELL THERAPY," filed December 8, 2009, both of which are incorporated herein by reference in their entireties.

[0002] This invention relates to a method for culturing cells, and more particularly to cell culture for cell therapy. [Background technology]

[0003] Cell culture is a major factor contributing to the cost and effort of cell therapy. Current methods make the cell culture process time-consuming and expensive. To generate large quantities of cells, a multi-step process is typically performed outside the body. At the earliest stage, the desired cells are a relatively small population within a cell population placed in a cell culture device. At this stage, the cell population contains a source of the desired cells (e.g., peripheral blood mononuclear cells), feeder cells that stimulate the growth of the desired cells, and / or antigen-presenting cells. Because the cells remain relatively undisturbed, culture devices and methods that typically do not disturb the medium in which the cells reside are preferred. Examples of such devices include standard tissue culture plates, flasks, and bags. Broadly speaking, culture proceeds in several steps: allowing the cell population to consume a medium containing growth substrates such as glucose, removing the spent medium, replacing the spent medium with fresh medium, and repeating the process until the desired quantity of the desired cells is obtained. As the desired cell population grows and requires another growth surface, the cell population is often transferred to another device and a new production step is initiated. However, with conventional methods, as the cell population on the growth surface increases, the growth rate of the desired cell population slows. As a result, producing a significant population of the desired cells requires considerable time and effort.

[0004] The traditional method for generating T lymphocytes with antigen specificity for Epstein-Barr virus (EBV-CTL) is one example of the laborious nature of this process. The traditional method for optimal expansion of EBV-CTL uses standard 24-well tissue culture plates, each well of which contains a cell-bearing surface area of ​​2 cm. 2 and the medium volume is 1 ml / cm due to gas transfer requirements. 2 The culture process begins with the establishment of a cell population of PBMCs (peripheral blood mononuclear cells) in the presence of irradiated antigen-presenting cell lines (which may also be lymphoblastoid cell lines (LCLs)).6 pieces / cm 2 , irradiated antigen-presenting cells 2.5 x 10 4 pieces / cm 2 surface density (i.e., growth surface 1 cm 2 The ratio of EBV-CTLs to the number of cells per 1000 cells was set to approximately 40:1. This resulted in an increase in the number of EBV-CTLs in the cell population. After 9 days, approximately 2.5 x 10 EBV-CTLs were detected. 5 pieces / cm 2 EBV-CTLs are again selectively expanded in the presence of irradiated antigen-presenting LCLs at a minimum surface density of 1000 μg / cm2, a new surface density ratio of 4:1. The medium volume is limited to a maximum of 1 ml / cm2 of growth area to allow oxygen, which limits growth solutes such as glucose, to reach the cells. 2 As a result, the maximum achievable EBV-CTL surface density is approximately 2 × 10 6 pieces / cm 2 Therefore, the maximum cell proliferation in one week is approximately 8-fold (i.e., 2 × 10 6 pieces / cm 2 2.5 x 10 5 pieces / cm 2 The mean cell density (m / s) of EBV-CTLs is less than 1 / 2 of the mean cell density (m / s) divided by 1 / 2. To continue expanding EBV-CTLs, the EBV-CTLs must be transferred to another 24-well plate once a week for antigen restimulation, and the medium and growth factors in each well of the 24-well plate must be replaced twice a week. With conventional methods, the rate of expansion of the EBV-CTL population slows as the EBV-CTL surface density reaches the maximum possible number per well. Therefore, these procedures must be repeated over a long production period, typically 4–8 weeks, to obtain sufficient numbers of EBV-CTLs for cell injection and quality control measures such as sterility testing, identity testing, and efficacy testing.

[0005] The culture of EBV-CTLs is just one example of the complex cell production process inherent in cell therapy. There is a need for more practical cell culture methods for cell therapy that can shorten the production time, while at the same time reducing the production costs and labor.

[0006] We have devised a new method that can increase the population growth rate throughout the entire production process, thereby reducing the effort and time required to produce cells.

[0007] Typically, primary nonadherent cells such as antigen-specific T cells, natural killer cells (NK cells), regulatory T cells (Tregs), tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (TILs), and pancreatic islets are targeted for generation. Many generation processes typically rely on other cell types to expand the population of desired cells (often called effector cells) in coculture conditions, stimulating their proliferation and antigen specificity. The cells used in this coculture are typically called feeder cells and / or antigen-presenting cells. In some cases, such as TIL generation, the coculture is expanded to expand the population of desired cells in the absence of feeder cells or antigen-presenting cells. Generation of antigen-presenting cells and / or feeder cells in the absence of effector cells is also widespread. In other cases, the culture is intended to maintain the health of the cell population rather than expand the population itself, such as in pancreatic islet culture for the treatment of diabetes. Therefore, the culture equipment and generation processes for cell culture in adoptive cell therapy must address many potential generation applications.

[0008] For adoptive cell therapy to be useful on a wide scale, the cell production process needs to be greatly simplified and inexpensive, but current production equipment and methods do not allow this. A simple explanation for why is as follows:

[0009] The devices currently relied upon heavily in the field of adoptive cell therapy are static cell cultures, i.e., cell culture plates, flasks, and gas-permeable bags. These static devices are intended to allow cells to remain in close proximity to one another during culture, facilitating exchange between co-cultures and / or allowing non-co-cultures to remain physically quiescent. As those skilled in the art will appreciate, a physically undisturbed state is advantageous for a variety of biological reasons. Furthermore, static cell culture devices are uncomplicated and do not require the constant use of auxiliary equipment during operation to perfuse medium or gas through the device, to agitate the device by sparging, stirring, or vibrating, and / or to prevent cells from settling to the bottom of the device. Thus, static devices are compatible with standard laboratory and cell culture equipment, such as incubators, and require minimal or no reliance on auxiliary equipment. While static devices have the advantages described above, they also have inherent problems that hinder the efficient and practical generation of cells for adoptive cell therapy.

[0010] Among these particular problems is the limitation of the height of the medium above the growth surface, which ranges from approximately 0.3 cm for plates and flasks to 2.0 cm for gas-permeable bags, as recommended by the manufacturer. Thus, plates and flasks require a medium volume to growth area ratio of 0.3 ml / cm. 2 and gas-permeable bags are limited to 2.0 ml / cm 2 The combined design limitations of plates, flasks, and bags are the current experimental designs used in the field of adoptive cell therapy. These experimental designs allow for cell densities between 0.5 and 2.0 x 10 6 The narrow range of cells / ml is essential to start a culture, with at least 0.5 × 10 6 pieces / cm 2These limitations create problems that make cell production for adoptive cell therapy impractical, including the excessive amount of equipment required during the process, the excessive labor required to maintain the cultures, the high risk of contamination, and / or the length of time it takes to produce the cells. Bags present a unique problem in that cells are disturbed from their resting position and transported into the medium during routine manipulation of the bag.

[0011] Alternative devices to plates, flasks, and bags have been introduced in co-pending U.S. Patent Application No. 2005 / 0106717 A1 to Wilson et al. (hereinafter referred to as Wilson '717) and U.S. Patent Application No. 2008 / 0227176 A1 to Wilson (hereinafter referred to as Wilson '176), and alternative methods of culture have been introduced in the parent applications, which describe particularly significant improvements to cell production processes in the field of adoptive cell therapy. Wilson '717 describes various innovative gas-permeable devices that allow culture methods to be performed at a vertical scale beyond the limited medium height and medium volume to growth area ratio limitations of plates and flasks, and that allow bags to use physical space more efficiently. Wilson '176 builds on Wilson '717 by allowing for a larger growth area in a given physical space. The parent application describes discoveries that allow for more efficient co-cultivation of cells commonly used in the field of adoptive cell therapy, including teachings that deviate from prior art limitations on cell areal density to provide a wide range of unexpected advantages.

[0012] The present invention builds upon the parent applications and includes new discoveries that further improve the efficiency and practicality of cell generation, particularly for the field of adoptive cell therapy, and builds upon Wilson '717 and Wilson '176, enabling various novel methods described herein. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] U.S. Patent Application No. 2005 / 0106717 [Patent Document 2] U.S. Patent Application No. 2008 / 0227176 Summary of the Invention

[0014] It has been discovered that cell therapy cell production can be accomplished in less time and more economically than currently possible by using a staged production process in which unconventional conditions can be periodically re-established during the production process. Among the unconventional conditions are the desired cell surface density (i.e., cells / cm). 2 These include reducing the cell size, achieving new ratios of cells of interest to antigen-presenting cells or feeder cells, and using growth surfaces made of gas-permeable materials with a high surface area to medium volume ratio.

[0015] Embodiments of the present invention relate to improved methods for culturing cells for cell therapy applications, including methods that reduce the time, cost, and effort required to produce desired numbers of cells of interest by using various novel methods that allow the desired cell population to maintain a higher proliferation rate throughout the production process compared to conventional methods.

[0016] One aspect of this invention is to perform a culture process in stages, with conditions established at the beginning of one or more stages that allow the desired cell population to expand at a rate greater than currently possible. At least one stage of the culture, and preferably substantially all stages, initial conditions are established, such as placing the desired cells at an unprecedented low surface density on a non-gas-permeable or gas-permeable growth surface and achieving an unprecedented ratio of desired cells to antigen-presenting cells (and / or feeder cells). Using novel embodiments of this aspect of the invention, the desired cell population can be doubled many times in a shorter time than previously possible with conventional methods, thereby reducing production time.

[0017] Another aspect of the invention is to perform a culture process in stages, with conditions established at the beginning of one or more stages that result in a growth rate of the desired cell population that exceeds currently possible. During at least one stage of the culture, and preferably at most all stages, the desired cells are placed on a growth surface made of a gas-permeable material, and conditions are established that result in an unprecedentedly high medium volume to growth area ratio. Using novel embodiments of this aspect of the invention, the desired cell population can be doubled many times in a shorter time than previously possible with conventional methods, thereby reducing production time.

[0018] Another aspect of this invention is to carry out the culture process in several stages, with conditions at each stage designed to grow the desired cell population at a rate greater than currently possible. At least one stage of the culture, and preferably at most all stages, the desired cells are grown at an unprecedentedly low surface density (i.e., cells / cm). 2 ) onto a growth surface made of a gas-permeable material, and initial conditions are established, including an unprecedented ratio of antigen-presenting cells (and / or feeder cells) to cells of interest, and an unprecedented high ratio of medium volume to growth surface area. Using novel embodiments of this aspect of the invention, the desired cell population can be doubled many times in a shorter time than previously possible with conventional methods, thereby reducing production time.

[0019] We have discovered an alternative cell culture method that builds upon the disclosures of the parent application while taking a different direction from traditional methods in the field of adoptive cell therapy to make the process of culturing and producing cells more practical and cost-effective than current methods.

[0020] In one embodiment, the present invention uses a gas-permeable cell culture device for culturing cells, and the cells are cultured at a surface density (cells / cm) when present in the gas-permeable device. 2) and cell density (cells / ml) can be reduced to levels lower than those achieved by conventional methods.

[0021] In another embodiment, the invention uses a gas permeable cell culture device for culturing cells, replacing the need to count cells to determine how many cells are in culture at any given time by taking solute samples in the medium and using them to estimate the population in the culture at any given time.

[0022] In another embodiment of the invention using a gas permeable cell culture device for culturing cells, the ratio of medium volume to growth surface area is increased to reduce the frequency of feeding after the culture is initiated compared to current methods, or to eliminate the need to feed the culture altogether.

[0023] In another embodiment, the invention utilizes gas permeable cell culture devices for culturing cells that have a larger medium volume to growth surface area ratio to allow the cell population to remain viable for a longer period of time after reaching maximum cell population.

[0024] In another embodiment of the present invention, a gas-permeable cell culture and harvesting device is disclosed that allows for the reduction of culture medium volume without cell loss, the concentration of cells without the need for centrifugation, and the increase of cell density prior to removal of cells from the device.

[0025] In another embodiment of the invention, a method for using novel gas permeable cell culture and cell harvesting devices is disclosed that allows an operator to reduce the medium volume in a culture without cell loss, minimizing the need to increase the number of devices in the culture when attempting to feed the culture.

[0026] Another embodiment of this invention, which uses a gas-permeable cell culture device for culturing cells, discloses a method for rapidly generating CAR T cells and improving their killing ability by using APCs during culture.

[0027] As another embodiment of the present invention using gas permeable cell culture devices for culturing cells, the method of the present invention can be scaled linearly in direct proportion to increases in growth surface area. [Brief explanation of the drawings]

[0028] The present invention will be more fully understood upon consideration of the following detailed description of various embodiments of the invention in conjunction with the accompanying drawings. [Figure 1A] It is shown that the antigen-specific T cell population in Example 1 undergoes at least seven cell doublings in the first seven days after initial stimulation. [Figure 1B] 1 shows data showing the magnitude of expansion of T cell populations within cell populations over time as determined by tetramer analysis for Example 1. [Figure 1C] In Example 1, it is shown that the rate of expansion of the antigen-specific T cell population slows over a 23 day period. [Figure 2] 1 is a table showing the discrepancy between the possible expansion and the observed fold expansion of antigen-specific T cells in Example 1. [Figure 3A] FIG. 1 shows the presence of antigen-specific T cells after stimulation in Example 2. [Figure 3B] In Example 2, we show the expansion of the antigen-specific T cell population when the surface density is reduced from 1 x 106 / cm2 to 3.1 x 104 / cm2 while maintaining a 4:1 ratio of antigen-specific T cells to antigen-presenting cells. [Figure 3C] In Example 2, the expansion of the antigen-specific T cell population is shown when the surface density is reduced from 1 x 106 / cm2 to 3.1 x 104 / cm2 in the presence of a constant population of antigen-presenting cells. [Figure 4]Continuing the process described in Figure 3, we present examples of results that can be obtained, and also demonstrate that when cells of interest require other cellular support, population growth can be initiated at unprecedentedly low surface densities, as long as the cells of interest are adequately supplied with feeder cells and / or antigen-presenting cells. [Figure 5] This is a histogram showing the reproducibility of the degree of expansion of the target cell population by initiating cultures at three different surface densities (number of CTLs / cm2). [Figure 6] 1 shows a cross-sectional view of the gas permeability test fixture used to obtain the data. [Figure 7A] 1 shows the proliferation curves of antigen-specific T cells produced according to the present invention compared with those of a conventional method, as performed in Example 5. [Figure 7B] For Example 5, cell viability, as determined by flow cytometric forward scatter versus side scatter analysis, was shown to be significantly higher in antigen-specific T cells generated according to the present invention compared to conventional methods. [Figure 7C] For Example 5, it is shown that cell viability determined by Annexin-PI 7AAD was significantly higher in antigen-specific T cells produced according to the present invention compared to conventional methods. [Figure 7D] For Example 5, the superior proliferation of cells produced by the novel method of this invention, as determined by daily flow cytometry analysis of CFSE-labeled cells, shows that the cells have the same intrinsic proliferation rate as cells cultured using conventional methods, confirming that cell killing results in an increased rate of cell proliferation. [Figure 8A] This demonstrates how EVB-CTL can be expanded beyond what is possible with conventional methods, without the need for medium replacement. [Figure 8B] The culture conditions of Example 6 demonstrate how they do not alter the final cell product, as assessed by Q-PCR for EBER. [Figure 8C]The culture conditions of Example 6 demonstrate how they do not alter the final cell product, as assessed by Q-PCR for the B cell marker CD20. [Figure 9] Here we provide an example where we have experimentally demonstrated that the proliferation of the AL-CTL population cannot be initiated when the cumulative surface density of the target cells and antigen-presenting cells (in this case, AL-CTL cells and LCL cells combined to form a cell population with a surface density of 30,000 cells / cm2) is very low. [Figure 10A] Data from Example 8 are presented that show how two novel methods of culturing cells produce more cells than conventional methods over a 23 day period. [Figure 10B] Photographs of cells cultured in the test fixture in Example 8 are shown. [Figure 10C] In Example 8, it is shown that the two novel culture methods and the conventional method all produce cells with the same phenotype. [Figure 10D] For Example 8, representative cultures are shown in which T cells stimulated with EBV peptide epitopes from EBV LMP1, LMP2, BZLF1, and EBNA1 and stained with HLA-A2-LMP2 peptide pentamer staining demonstrated similar frequencies of peptide-specific T cells. [Figure 10E] For the novel method of Example 8 and the conventional method, the cells maintained their cytolytic activity and specificity, and killed autologous EBV-LCL with reduced killing of HLA-mismatched EBV-LCL, as assessed by the 51Cr release assay. [Figure 11] 1 shows a graphical representation of the population growth of a desired cell on a growth surface under a conventional scenario compared to the population growth of a desired cell type using one aspect of the present invention. [Figure 12] Examples of the advantages that can be obtained by utilizing growth surfaces constructed from gas permeable materials and unprecedentedly high medium volume to growth area ratios of 1 or 2 ml / cm2 or greater are shown. [Figure 13]1 is a graphical representation of a novel method of population expansion of a desired cell on a growth surface under a conventional scenario compared to the population expansion of a desired cell type in one embodiment of the present invention, where the surface density of cells at the end is much greater than the conventional surface density. [Figure 14] Another novel method of cell engineering is presented, which offers additional advantages over conventional methods. [Figure 15] To demonstrate the power of the new method and why it is useful to adjust the fabrication protocol to fully exploit the efficiency at various stages, a comparison of each fabrication method is shown in Figure 14. [Figure 16] An example is provided of how the production protocol can be adjusted in a novel manner to gain efficiency as production progresses. [Figure 17A] A representative spreadsheet of experimental conditions and results at 1.0E+06 particles / cm2 is shown. [Figure 17B] A representative spreadsheet of experimental conditions and results at 0.5E+06 particles / cm2 is shown. [Figure 17C] A representative spreadsheet of experimental conditions and results at 0.25E+06 particles / cm2 is shown. [Figure 17D] A representative spreadsheet of experimental conditions and results at 0.125E+06 particles / cm2 is shown. [Figure 17E] A representative spreadsheet of experimental conditions and results for 0.0625E+06 particles / cm2 is shown. [Figure 18] Figures 17A-17E show a comparison of fold population expansion versus surface density for each of the experimental conditions detailed. [Figure 19A] A representative spreadsheet of experimental conditions and typical results for cultures of K562 cells under comparable starting conditions except for glucose concentration is shown. [Figure 19B] Cell population growth over 11 days under two glucose starting conditions is shown. [Figure 19C]The glucose reduction rate under each culture condition is shown. [Figure 19D] The glucose consumption rate under each culture condition is shown. [Figure 19E] Shown is an overlay of the predicted cell population using formula calculations and manual cell counts for cultures initiated at a glucose concentration of 240 mg / dl. [Figure 19F] Shown is an overlay of the predicted cell population using formula calculations and manual cell counts for cultures initiated at a glucose concentration of 240 mg / dl. [Figure 20] 1 is a graphical representation of population growth normalized to growth surface under various substrate feeding conditions. [Figure 21] 1 shows a spreadsheet summarizing conditions on days 0, 9, and 16 for an experiment demonstrating that glucose decline can be used as a surrogate measure of cell population. [Figure 22A] 1 shows a cross-sectional view of one example of an inventive embodiment of a cell culture and cell harvesting device 1000 configured to perform the disclosed novel cell culture methods and / or novel cell harvesting methods. [Figure 22B] The cell culture and cell harvesting device 1000 is shown in an initial state of static culture at the beginning of any given cell production stage of culture. [Figure 22C] 1 shows a cell culture and cell harvesting device 1000 prepared to harvest cells in a reduced volume of medium. [Figure 22D] 10. The process of reorienting the cell culture and cell collection device 1000 from its original horizontal cell culture position to a position deflected by an angle 1026 for transferring cell collection medium 1024 is shown. [Figure 23A] The conditions for assessment A at the beginning of the culture and as the culture progressed are shown. [Figure 23B] The conditions for assessment B at the beginning of the culture and as the culture progressed are shown. [Figure 23C] The conditions for assessment C at the beginning of the culture and as the culture progressed are shown. [Figure 23D] Shown are the total viable cells in the culture at various time points during the culture. [Figures 23E1-23E3] The percentage of CAR T cell expression at the beginning and end of culture is shown. [Figure 23F] The total fold expansion of CAT T cells in culture is shown. [Figure 23G] FIG. 1 shows that the predicted viable cell population in assessment A represented the manually counted cell population. [Figure 23H] The ability of cells from condition A and condition B to kill tumor cells expressing PSCA is shown. [Figure 24A] Side-by-side comparison of the expansion of CAR T cell populations with specificity for PSCA. [Figure 24B] Side-by-side comparison of the expansion of CAR T cell populations with specificity for Muc1. [Figure 24C] 1 is a graph of CAR T cell population expansion. [Figure 24D] Graph of Muc1 cell population expansion. [Figure 24E] Population growth curves for three gas-permeable culture devices with different growth areas are shown. [Figure 24F] The population growth of the curves in Figure 24E is shown after normalization to surface density. DETAILED DESCRIPTION OF THE INVENTION

[0029] definition adherent cell cell attached to a growth surface Antigen-presenting cell (APC): A cell that acts as a trigger for target cells to respond to a specific antigen. CTL: Cytotoxic T cell Cell density: the ratio of the number of cells to the unit volume of medium (cells / ml) Cells of interest: Cells of a particular type whose quantity is intended to be expanded or harvested through a production process. Generally, cells of interest are nonadherent, and examples include regulatory T cells (Tregs), natural killer cells (NKs), tumor-infiltrating lymphocytes (TILs), primary T lymphocytes, and cells with a broad range of antigen specificities, as well as many others (all of which can be genetically engineered to alter their function, in vivo persistence, and safety). Cells required for therapeutic applications can be expanded using feeder cells and / or antigen-presenting cells. These include PBMCs, PHA blasts, OKT3 T and B blasts, LCLs, and K562 (natural or genetically engineered to express antigens and / or epitopes and costimulatory molecules such as 41BBL, OX40, CD80, CD86, HLA, and many others), which may or may not be pulsed with peptides or other relevant antigens. EBV: Epstein-Barr virus EBV-CTL: specifically recognized EBV-infected cells or cells that express or present EBV-derived peptides via their T cell surface receptors. EBV-LCL: B lymphoblastoid cell line transformed by Epstein-Barr virus. Feeder cells: Cells that act to quantitatively expand target cells. Depending on the environment, antigen-presenting cells can also act as feeder cells. Growth surface: The area of ​​the culture device on which the cells rest. Start of culture: Generally refers to the conditions at the beginning of a culture process or production cycle. Medium exchange: Synonymous with providing nutrients to cells, it is usually the process of replenishing old medium with fresh medium. PBMC: Peripheral blood mononuclear cells extracted from peripheral blood, which are a source of some of the cells of interest and can act as feeder cells. Responder (R): A cell that responds to a stimulator cell. Static cell culture: A method of culturing cells in a medium that is not agitated or mixed except when the culture device is moved for routine handling and / or the cells are periodically fed with fresh medium or other fluids. Generally, the medium in static culture is quiescent. The medium is not subjected to forced movement such as occurs in perfusion systems (where the medium is constantly moving within the vessel), vibration systems in which the culture device is physically vibrated to move the medium, agitation systems (where a stir bar moves within the device to agitate the medium and cells), or any other mechanism or device used to move and mix the medium during culture. The cells settle to the growth surface in the device and remain there undisturbed except for occasional feeding periods. During feeding, the culture is provided with fresh medium by first removing the medium and then adding medium, or by adding medium without removing medium, or by removing the medium and cells, transferring the medium and cells to a new device, and adding fresh medium to the device. Pumps to assist the feeding process are common. For example, gas-permeable cell culture bags typically rely on gravity or pumps to move fluids within a closed system. The majority of the culture period is during which the cells and medium reside in a quiescent, undisturbed state. This invention is directed to a method for quiescent cell culture. Stimulation: The effect that antigen-presenting cells and / or feeder cells have on cells of interest. Stimulator (S): A cell that influences responder cells. Surface Density: The amount of cells per unit area of ​​the growth surface in the device on which the cells rest. Suspension cells: Cells that do not need to be attached to a growth surface; synonymous with non-adherent cells.

[0030] A series of experiments was conducted to discover a novel method for easily generating desired populations of cells for adoptive T cell therapy, opening the door to more efficient cultivation of cells for cell therapy applications. Numerous illustrative examples and various aspects of this invention are described to show how the benefits of reduced production time and labor compared to conventional methods are realized.

[0031] Example 1: Demonstrating the limitations of traditional methods The data in this example are based on a standard 24-well tissue culture plate (i.e., a surface area of ​​2 cm per well). 2 ) with a volume of 2 ml of medium per well (i.e., a medium height of 1.0 cm, a medium volume to surface area ratio of 1 ml / cm 2 ) to demonstrate the limitations of conventional culture methods for producing EBV-CTLs.

[0032] First stage of culture, day 0: A cell population (approximately 1 × 10 ) containing PBMCs from normal donors and antigen-presenting autologous gamma-irradiated (40 Gy) EBV-LCLs at a ratio of 40:1 (PBMC:LCL) was cultured. 6 Expansion of the EBV-CTL population was initiated by culturing 1000 cells / ml in RPMI 1640 supplemented with 45% Click medium (Irvine Scientific, Santa Ana, CA), 2 mM GlutaMAX-I, and 10% FBS at a medium volume to growth area ratio of 1 ml / cm. 2 The surface density is approximately 1×10 6 cells / cm 2 was set to.

[0033] Second stage of culture, days 9 to 16: On day 9, EBV-CTLs were harvested from the cell population generated in the first stage, and EBV-CTLs were collected at 0.5 × 10 6 pieces / cm 2 and resuspended in fresh medium at a surface density of 0.5 x 10 CTLs at a CTL:LCL ratio of 4:1 (surface density 0.5 x 10 CTLs). 6 pieces / cm 2 :1.25×10 5 pieces / cm 2 ), and restimulated with irradiated autologous EBV-LCL. On day 13, 1 ml of the 2 ml of medium in each well of a 24-well plate was removed and replaced with 1 ml of fresh medium containing recombinant human interleukin-2 (IL-2) (50 U / mL) (Proleukin, Chiron, Emeryville, CA).

[0034] Stage 3 of culture, days 17–23: Stage 2 conditions were repeated with the addition of IL-2 twice a week, and culture was terminated on day 23. Although the culture was terminated, additional culture stages could have been continued, mimicking stages 2 and 3.

[0035] Cell lines and tumor cells used as target cells in the cytotoxicity assay: BJAB (B-cell lymphoma) and K562 (chronic erythroleukemia) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). All cell cultures were maintained in RPMI 1640 medium (GIBCO-BRL, Gaithersburg, MD) containing 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 25 IU / mL penicillin, and 25 mg / mL streptomycin (all from BioWhittaker, Walkersville, MD). Cells were incubated at 37°C in 5% CO. 2 The specimen was kept in a humid atmosphere containing

[0036] Immunomarker diagnostics: Cells were stained with phycoerythrin (PE), fluorescein isothiocyanate (FITC), periodin chlorophyll protein (PerCP), and allophycocyanin (APC)-conjugated monoclonal antibodies (MAbs) against CD3, CD4, CD8, CD56, CD16, CD62L, CD45RO, CD45RA, CD27, CD28, CD25, and CD44 (obtained from Becton-Dickinson, Mountain View, CA, USA). EBV-CTL precursor frequencies were quantified using PE-conjugated tetramers (Baylor College of Medicine) and APC-conjugated pentamers (Proimmune, Oxford, UK). For cell surface and pentamer staining, 10,000 and 100,000 biological events were collected on a FACSCalibur flow cytometer, respectively, and data were analyzed using Cell Quest software (Becton Dickinson).

[0037] CFSE labeling to measure cell division: To determine doubling rate, 2 x 10 7 PBMCs or EBV-specific CTLs (EBV-CTLs) were washed twice and resuspended in 850 μl of 1× phosphate-buffered saline (PBS) containing 0.1% fetal bovine serum (Sigma-Aldrich). Prior to staining, carboxyfluorescein diacetate, succinimidyl ester (CFSE) (10 mM in dimethyl sulfoxide) (Celltrace tm An aliquot of CFSE cell proliferation kit (C34554, Invitrogen) was thawed and diluted 1:1000 in 1x PBS, and 150 μl of the dilution was added to the cell suspension (labeling concentration was 1 μM). Cells were incubated with CFSE at room temperature for 10 minutes. Subsequently, 1 ml of FBS was added to the cell suspension, followed by incubation at 37°C for 10 minutes. Afterwards, cells were washed twice with 1x PBS, counted, and stimulated with antigen as described above.

[0038] Annexin V-7-AAD staining: To determine the percentage of apoptotic and necrotic cells in our cultures, we used the annexin V-7-AAD staining method according to the manufacturer's instructions (BD Pharmingen tm Staining with Annexin-7-AAD was performed according to the protocol described in the EBV-CTLs section (#559763, San Diego, CA). Briefly, EBV-CTLs from 24-well plates or G-Rex were washed with cold PBS and diluted to 1 × 10 in 1 × Binding Buffer. 6 The cells were resuspended at a cell concentration of 100 / ml and stained with Annexin V-PE and 7-AAD for 15 minutes at room temperature (25°C) in the dark. Following incubation, the cells were immediately analyzed by flow cytometry.

[0039] Chromium Release Test: As we previously mentioned, a standard 4-hour 51 The cytotoxicity of EBV-CTLs was evaluated in a Cr release assay. We used autologous HLA class I and II mismatched EBV-transformed lymphoblastoid cell lines (EBV-LCL) as target cells to measure MHC-restricted and non-restricted killing, and the K562 cell line to measure natural killer activity. Chromium-labeled target cells cultured in medium alone or in 1% Triton X-100 were used to measure spontaneous killing. 51 Cr free and max 51 Cr release was determined for each well. The average percentage of specific lysis for triplicate wells was calculated as follows: [(test counts - spontaneous counts) / (maximum counts - spontaneous counts)] x 100.

[0040] Enzyme-linked immunospot (ELIspot) assay: ELIspot assay was used to quantify the frequency and function of T cells that secreted IFNγ in response to antigen stimulation. CTL lines grown in 24-well plates or in G-Rex were stimulated with irradiated LCL (40 Gy) or a pepmix of LMP1, LMP2, BZLF1, and EBNA1 (diluted to 1 μg / ml) (JPT Technologies GmbH, Berlin, Germany), or with the EBV peptides HLA-A2 GLCTLVAML=GLC, HLA-A2 CLGGLLTMV=CLG, HLA-A2-FLYALALLL=FLY, and HLA-A29 ILLARLFLY=ILL (Genemed Synthesis, Inc., San Antonio, TX) diluted to a final concentration of 2 μM. CTL alone served as a negative control. CTLs were cultured at 1 × 10 in ELI spotting medium [RPMI 1640 (Hyclone, Logan, UT) supplemented with 5% human serum (Valley Biomedical, Inc., Winchester, VA) and 2 mM L-glutamine (GlutaMAX-I, Invitrogen, Carlsbad, CA)]. 6 The cells were resuspended at 1000 μg / ml.

[0041] A 96-well filtration plate (Millipore Multiscreen, #MAHAS4510, Bedford, MA) was coated with 10 μg / mL anti-IFN-γ antibody (Catcher-mAB91-DK, Mabtech, Cincinnati, OH) overnight at 4°C, then washed and blocked with ELI spotting medium for 1 hour at 37°C. Responder and stimulator cells were cultured on plates for 20 hours, then the plates were washed and incubated with a supplemental biotin-conjugated anti-IFN-γ monoclonal antibody (Detector-mAB(7-B6-Biotin), Mabtech), followed by incubation with an avidin:biotinylated horseradish peroxidase mixture (Vectastain Elite ABC Kit (Standard), #PK6100, Vector Laboratories, Burlingame, CA), and then developed with AEC substrate (Sigma, St. Louis, MO). Each culture condition was performed in triplicate. Plates were sent to Zellnet Consulting, New York, NY, for evaluation. Spot-forming units (SFC) versus input cell number was plotted.

[0042] Statistical analysis: In vitro data were expressed as mean ± 1 SD. Student's t-test was used to determine statistical significance between samples, and P < 0.05 indicated significant differences.

[0043] Under these culture conditions, the antigen-specific T cell population undergoes at least seven cell doublings within the first 7 days after initial stimulation, as shown in Figure 1A. Therefore, a 128-fold T cell expansion (measured by multiplying the frequency of antigen-specific T cells by the total number of cells in the cell population) is expected per week. The frequencies of tetramer-positive cells after the first, second, and third stimulations are shown in Figure 1B. On day 0, the frequencies of T cells reactive to the two EBV tetramers, RAK and QAK, were 0.02% and 0.01%, respectively. After a single stimulation on day 0, tetramer-positive T cells in the cell population increased from 0.02% and 0.01%, to 2.7% and 1.25%, respectively, by day 9. Thus, a 135-fold and 125-fold increase in the proportion of antigen-specific tetramer-positive T cells present in the cell population, as measured by RAK and QAK, was achieved. Also, after a single stimulation in the first stage of culture on day 0, by day 9, a 1.1-fold increase (not shown) in the surface density of cells in the cell population was observed (approximately 1.1 × 10 6 pieces / cm 2 (Figure 1C shows that the antigen-specific T cell population expanded approximately 280-fold during the first stage of culture, although the overall increase in cell numbers was small because the majority of cells in the PBMC composition were not specific for the stimulating antigen. Unfortunately, although the number of cell doublings measured by CSFE was the same during the second and third stages of culture, this rate of expansion of antigen-specific T cells was not sustained during the second and third stages of culture, with only 5.7 expansions and 4.3 expansions during the second and third stages. The table in Figure 2 demonstrates the discrepancy between the potential expansion and the observed fold expansion of antigen-specific T cells (n = 3).

[0044] Example 1 shows that the time it takes to generate cells of interest generally slows after the first approximately week of generation, as the population growth rate of the cells of interest slows in subsequent stages.

[0045] Example 2: By reducing the cell surface density of the cells of interest at the start of any given stage or stages in the culture, a reduction in the time required to expand the population of the cells of interest can be achieved.

[0046] We hypothesized that the slower expansion rate of the desired cell population after the second T cell stimulation compared to the first stimulation may be due to restrictive cell culture conditions, resulting in activation-induced cell death (AICD). For example, referring to Figure 3A, in the first stimulation, the EBV antigen-specific T cell component of PBMCs was at most 2% of the population, and therefore the plating density of antigen-specific responder T cells was 2 × 10 4 / cm 2 The remaining PBMCs act as non-proliferating feeder cells (visible as CFSE-positive cells in Figure 3A), maintaining optimal cell-cell contact and allowing proliferation of antigen-specific CTLs. In contrast, during the second stimulation on day 9, the majority of T cells are antigen-specific, and although the total cell density of the composition is approximately the same, the proliferating cell density is 50 to 100 times higher. Consequently, upon restimulation, the majority of cells proliferate, which can rapidly consume and deplete the nutrient and O2 supply.

[0047] To determine whether limiting culture conditions were causing suboptimal T cell growth rates, we measured the proliferation of activated T cells seeded at lower cell densities, as described previously in Example 1.

[0048] As shown in Figure 3B, we cultured activated EBV-specific T cells in wells with a growth area of ​​2 cm. 2 The responder cells were seeded into standard 24-well plates at 1 × 10 cells per well. The responder cells were serially diluted to a surface density of 1 × 10 cells per well, maintaining a 4:1 ratio of responder to stimulator cells (R:S). 6 / cm 2 to 3.1 x 10 4 / cm 2 The range is set to 1.25×10 5 / cm2 Although a starting CTL surface density of 1.25 × 10 resulted in maximal CTL proliferation (4.7 ± 1.1-fold), further dilution slowed down the proliferation rate, as shown in Figure 3B. We attribute this limited effect of dilution to a lack of cell-cell contact, and therefore, we maintained a constant number of feeder cells (1.25 × 10). 5 / cm 2 EBV-LCLs were seeded at a surface density of 1 x 10 6 to 3.1 x 10 4 EBV-CTLs were cultured at serial dilutions up to 1 × 10 and cell proliferation was examined over 7 days. As shown in Figure 3C, 1 × 10 6 / cm 2 The surface density of EBV-CTLs was 3.1 × 10, up from only 2.9 ± 0.8 times that of EBV-CTLs. 4 / cm 2 We observed a dramatic increase in CTL proliferation, with EBV-CTLs at a surface density of 1.7 to 34.7 ± 11-fold expansion. Importantly, this modification of culture conditions did not alter the function or antigen specificity of the cells (data not shown). Thus, the population of activated antigen-specific T cells can be expanded to a greater extent than conventional culture methods allow. Of note, the maximum surface density (1.7 to 2.5 × 10) activated after stimulation was 1.7 to 2.5 × 10. 6 / cm 2 ) was the same regardless of the starting surface density.

[0049] Thus, conventional culture conditions are limited, and the ratio of medium volume to growth surface area is limited to the conventional 1 ml / cm 2This indicates that it is necessary to increase the surface density of the target cell population beyond the surface density limit of conventional methods. Furthermore, by lowering the surface density of the target cell population at the start of any stage of culture to below that of conventional methods, the proliferation of antigen-specific CTLs can be improved by approximately 34-fold. This has a significant effect in cell therapy, where the amount of cells at the start of production is usually very limited. Because the growth rate of the population is dramatically increased compared to conventional surface densities, for example, a larger population of target cells can be achieved in a shorter time by distributing a limited amount of target cells over a wide surface area at a low surface density.

[0050] Example 3: A minimum surface density of a population of cells, including cells of interest and / or antigen presenting cells, allows for the expansion of a population of cells of interest seeded at very low surface densities.

[0051] Figure 4 shows an example of the results obtained by continuing the procedure described in Figure 3. The results also demonstrate that if the cells of interest require other cellular support, population expansion can be initiated at an unprecedentedly low surface density of the cells of interest, as long as the cells of interest are kept in a sufficient supply of feeder cells and / or antigen-presenting cells. In these experiments, we subsequently cultured approximately 1.0 × 10 cells of interest at an R:S ratio of 8:1. 6 pieces / cm 2 With an R:S ratio of 1:32, only about 3900 target cells / cm 2 We show how all cell populations with surface densities and R:S ratios between 0.01 and 0.10 were able to significantly expand the cells of interest to over 50 times the initial surface density, at which point we stopped the study.

[0052] Example 4: Unprecedented low surface density of target cells. The ability to repeat the production process in several steps, starting a step, expanding the population, ending the step, and repeating these conditions, has demonstrated the ability to obtain reproducible results.

[0053] As shown in Figure 5, three target cell surface densities (CTL / cm 2 ), the evaluation described in Example 3 was continued. Each particular seeding density was able to consistently achieve the same expansion fold. The implications of this will be discussed in more detail below as it relates to the ability to dramatically reduce production time for a given cell population.

[0054] Example 5: By culturing cells of interest on a growth surface constructed from a gas permeable material while increasing the ratio of medium volume to growth surface area, the number of population doublings of the cells of interest at a given stage of culture is increased compared to conventional methods, and the achievable surface density is increased.

[0055] Cell lines and tumor cells, immunological marker diagnosis, CFSE labeling, Annexin V-7-AAD staining, chromium release assay, enzyme-linked immunospot (ELIspot) assay, retroviral production and transduction into T lymphocytes, and statistical analysis were as described in Example 1.

[0056] The test fixtures (hereafter generally referred to as "G-Rex") were constructed as shown in FIG. 6. The bottom 20 of each G-Rex 10 was formed from a gas-permeable silicone membrane approximately 0.005 to 0.007 inches thick. Wilson's pending U.S. Patent Application No. 2005 / 0106717 A1 is one of many other sources of information using other gas-permeable materials and can be used to educate those skilled in the art on the geometry and features of gas-permeable cultureware and other useful properties that are advantageous for many embodiments of this invention. In this Example 3, the G-Rex (hereafter referred to as "G-Rex 40") was constructed as a 10 cm 2 The cells had a growth area of ​​1000 mm, on which a cell population (shown as item 30) was placed. The characteristics of this cell population varied throughout the experiments described therein. The medium volume (shown as item 40) was 30 ml unless otherwise indicated, with a density of 3 ml / cm. 2 This forms the ratio of medium volume to growth area.

[0057] Activated EBV-specific CTLs and irradiated autologous EBV-LCLs were cultured in G-Rex40 devices at a conventional CTL:LCL ratio of 4:1. EBV-CTLs were cultured at 5 × 10 5 pieces / cm 2 and seeded at a surface density of 1 ml / cm 2 The population growth rate of EBV-CTLs was compared to that of EBV-CTLs seeded at the same surface density in standard 24-well plates with a medium volume to growth area ratio of 5 × 10. After 3 days, as shown in Figure 7A (p = 0.005), without medium replacement, EBV-CTLs in G-Rex40 cells grew to 5 × 10 cells / well. 5 / cm 2 From the median, 7.9 × 10 6 / cm 2 (5.7 to 8.1 × 10 6 / cm 2 In contrast, EBV-CTLs cultured for 3 days in conventional 24-well plates expanded to a surface density of 5 × 10 by day 3. 5 / cm 2 From the median, 1.8 × 10 6 / cm 2 (1.7 to 2.5 × 10 6 / cm 2 In G-Rex40, the surface density could be further increased by supplementing the medium, whereas in 24-well plates, supplementing the medium or IL-2 did not increase the cell surface density. For example, after supplementing the medium or IL-2 on day 7, the surface density of EBV-CTL in G-Rex40 was 9.5 × 10 6 pieces / cm 2 (8.5×10 6 to 11.0 x 10 6 / cm 2 range) (data not shown).

[0058] To understand the mechanism behind the superior cell proliferation in the G-Rex device, we assessed the viability of OKT3-stimulated peripheral blood T cells on day 5 of culture using forward and side scatter analysis by flow cytometry. EBV-CTLs could not be assessed in this study due to the presence of residual irradiated EBV-LCL in the cultures, which interfered with the analysis. As shown in Figure 7B, cell viability was significantly higher in G-Rex40 cultures (89.2% viability in G-Rex40 vs. 49.9% viability in 24-well plates). We then analyzed each culture daily for 7 days using annexin-PI 7AAD to distinguish between live and apoptotic / necrotic cells. As shown in Figure 7C, we observed consistently lower viability of T cells expanded in 24-well plates compared to those in G-Rex. These data indicate that improved cumulative survival of proliferating cells contributed to the increased number of cells in the G-Rex device compared to 24-well plates.

[0059] To determine whether the increased number of cell divisions in the G-Rex versus 24-well plates also contributed to the increase, T cells were labeled with CFSE on day 0 and split between G-Rex40 fixtures containing 40 ml of medium and 24-well plates containing 2 ml of medium per well. Daily flow cytometry analysis showed no difference in the number of cell divisions from days 1 to 3. However, from day 3 onward, the population of desired cells cultured in the G-Rex40 continued to increase at a rate that exceeded the declining rate in the 2 ml wells, indicating that the 2 ml wells were reaching their limiting culture conditions, as shown in Figure 7D. Thus, the increased population of desired cells in the G-Rex40 test fixture was the result of a combination of reduced cell death and sustained proliferation relative to conventional methods.

[0060] Example 6: By using growth surfaces constructed from gas permeable materials with unprecedentedly high medium volume to growth surface area ratios, unprecedented surface densities of desired cells can be achieved during production while simultaneously reducing the need to feed the culture.

[0061] This was demonstrated using the G-Rex test fixture for the initiation and propagation of EBV:LCL. For this example, a 100 cm 2 The G-Rex2000 refers to the device described in Figure 8, except that it consists of a growth surface of 1000 mm and has a 2000 ml medium volume available. EBV-LCLs were cultured and expanded in the G-Rex2000 without altering the cell phenotype. The medium volume to surface area ratio was 10 ml / cm. 2 To achieve a surface density of 1 x 10 cells, add 1000 ml of complete RPMI medium. 5 pieces / cm 2 EBV-LCL were seeded in G-Rex2000 at 0.18 ml / cm for comparison. 2 To achieve a medium volume to surface area ratio of 100, 30 ml of complete RPMI medium was added to the plate at a surface density of 5 × 10. 5 pieces / cm 2 EBV-LCLs were seeded into T175 flasks using a 100% ethanol solution. As shown in Figure 8A, EBV-LCLs cultured in G-Rex2000 expanded more than those in T175 flasks without any manipulation or medium change. As shown in Figures 8B and 8C, this culture condition did not alter the final cell product, as assessed by Q-PCR for EBER and the B cell marker CD20.

[0062] Example 7: If sufficient feeder and / or antigen-presenting cells are not present at the beginning of the culture, the cells of interest may not grow. However, the cell population can be modified to include other types of cells that act as feeder and / or antigen-presenting cells to allow growth.

[0063] In the illustrative example shown in Figure 9, cells of interest and antigen-presenting cells were cultured at a very low cumulative surface density (in this case, AL-CTL and LCL cells combined at a surface density of 30,000 cells / cm). 2 We have experimentally demonstrated that the surface density of approximately 0.5 × 10 cells (forming a cell population of approximately 0.5 × 10 cells) is incapable of initiating the expansion of a population of AL-CTLs. However, we were able to make the same cell population viable by modifying its composition to include another cell type acting as a feeder cell. In this case, a surface density of approximately 0.5 × 10 cells was obtained. 6 pieces / cm 2 We evaluated three different forms of feeder layers of irradiated K562 cells, and in all cases, the AL-CTL population expanded from the initial cell population depicted in the first column of the histogram to a cell surface density of only 15,000 cells / cm. 2 4.0 x 10 in 14 days 6 pieces / cm 2 We also demonstrated that similarly favorable results can be achieved by expanding the LCL population rather than adding a third cell type. The large surface densities used for LCL or K562 were chosen arbitrarily to demonstrate that growth can be initiated using very low populations of the desired cells when the cell population contains an adequate number of feeder cells and / or antigen-presenting cells. When there is an insufficient supply of expensive and cumbersome feeder cells, the surface density can be reduced to 0.5 × 10 6 pieces / cm 2 Generally, and as we have shown, when antigen-presenting cells and / or feeder cells are present in a cell population, the combined surface density of the antigen-presenting cells or feeder cells and the cells of interest should be at least about 0.125 x 10 to form a surface density in the cell population sufficient to initiate proliferation of the desired cell population. 6 pieces / cm 2 Also, to continue growth beyond the limits of standard surface density, in this example, 4 ml / cm 2 A growth surface constructed from a gas permeable material was used with a medium volume to surface area ratio of .

[0064] Example 8: By lowering the surface density of the desired cells, changing the ratio of responder cells to stimulator cells, increasing the medium to growth area ratio, and periodically distributing the cells on a growth surface composed of a gas-permeable material in a low surface density culture, more desired cells can be produced in a shorter time when compared to other methods, simplifying the production process.

[0065] To further evaluate our ability to simplify and shorten the generation of cells of interest, we used the G-Rex test fixture for initiation and propagation of EBV-CTL. For this example, we used a 100 cm bottom plate. 2 The G-Rex500 refers to the device described in Figure 6, except that it consists of a growth area of ​​1000 ml and has a medium volume of 500 ml available.

[0066] For the initial stage of EBV-CTL generation, we used 1 × 10 6 / cm 2 Surface density (overall = 10 cm of G-Rex40) 2 10 distributed throughout the growing area of 7 PBMCs were seeded into G-Rex40 and stimulated with EBV-LCL at a 40:1 PBMC:EBV-LCL ratio. For CTL generation, this 40:1 ratio is preferred for the first stimulation to maintain the antigen specificity of responder T cells. The second stage of culture was initiated 9 days after the first stage, in which 1 × 10 PBMCs were seeded into G-Rex500 test fixtures. 7 To start the second stage of the culture, 200 ml of CTL medium was placed in the G-Rex500, and the ratio of medium volume to surface area at the start of the second stage was 2 ml / cm. 2 The medium height was adjusted to 2.0 cm above the growth surface. The surface density of the target cells at the start of the second stage was 1 × 10 CTL. 5 pieces / cm 2 and LCL 5×10 5 pieces / cm 2Using a surface density of antigen-presenting cells of 100 μg / cm2, an unconventional ratio of cells of interest to antigen-presenting cells was achieved. This second-stage surface density and R:S ratio consistently resulted in EBV-CTL proliferation for all donors screened. Four days later (day 13), IL-2 (50 U / ml final concentration) was added directly to the cultures in 200 ml of fresh medium, resulting in a medium volume to surface area ratio of 4 ml / cm2. 2 On day 16, cells were harvested and counted. The median surface area of ​​the resulting CTL was 6.5 x 10 6 / cm 2 (2.4×10 6 From 3.5 x 10 7 (range).

[0067] Compared to traditional experimental designs, the use of a growth surface made of gas-permeable material allows for a larger medium volume to surface area ratio (i.e., 1 ml / cm). 2 or higher) and keep the cell surface density low (i.e., 0.5 × 10 6 / cm 2 (below), allowing for a variable ratio of responder cells to stimulator cells (up to 4:1), resulting in shorter production times. Figure 10A shows the results of comparing this G-Rex approach of Example 8 with the use of the conventional method of Example 1 and the G-Rex approach described in Example 5. As shown, the conventional method required 23 days to generate the number of desired cells that could be generated in approximately 10 days using either G-Rex method. After 23 days, the G-Rex approach of Example 8 was able to generate 23.7 times more desired cells than the G-Rex method of Example 5 and 68.4 times more desired cells than the conventional method of Example 1. Furthermore, the cell surface density was increased to 7 x 10 6 / cm 2 Under the condition that the cultures were split when they reached a size greater than 100 μg / ml, the cells continued to divide from days 27 to 30 without the need for additional antigen-presenting cell stimulation.

[0068] Although CTLs were not clearly visible in G-Rex cells using a light microscope, clusters of CTLs could be observed visually or by inverted microscopy. The appearance of the cells on days 9, 16, and 23 is shown in Figure 10B. As shown in Figure 10C, culture in G-Rex cells did not alter the phenotype of the expanded cells. More than 90% of the cell population was CD3+ cells (96.7±1.7 and 92.8±5.6 cells in G-Rex and 24-well cultures, respectively), which were primarily CD8+ (62.2%±38.3 and 75%±21.7 cells in G-Rex and 24-well cultures, respectively). Evaluation of activation markers CD25 and CD27 and memory markers CD45RO, CD45RA, and CD62L demonstrated virtually no differences between EBV-CTLs expanded under each culture condition. Antigen specificity, as measured by ELISA spots and pentamers, was also unaffected by the culture conditions. Figure 10D shows representative cultures of T cells stimulated with EBV peptide epitopes from LMP1, LMP2, BZLF1, and EBNA1 and stained with HLA-A2-LMP2 peptide pentamer staining, demonstrating similar frequencies of peptide-specific T cells. Furthermore, expanded cells maintained their cytolytic activity and specificity, and as shown in Figure 10E, 51 As assessed by Cr release assay, autologous EBV-LCLs were killed (62% ± 12 and 57% ± 8 for G-Rex and 24-well plates at an E:T ratio of 20:1), whereas killing of HLA-mismatched EBV-LCLs was low (15% ± 5 and 12% ± 7 at 20:1).

[0069] Discussion of various new and improved cell generation methods for cell therapy: Examples 1-8 are provided to demonstrate to those skilled in the art how various conditions, including lowering the surface density of the desired cell population at the beginning of the production cycle, lowering the surface density ratio between responder and stimulator cells, constructing the growth surface from a gas-permeable material, and / or increasing the medium volume to growth surface area ratio, can facilitate and simplify cell production for research and cell therapy medical applications. While Examples 1-8 were directed to the production of antigen-specific T cells, these novel culture conditions can be applied to many important suspension cell types relevant to therapy (or required for proof-of-concept in pre-therapeutic mouse models), including regulatory T cells (Tregs), natural killer cells (NKs), tumor-infiltrating lymphocytes (TILs), primary T lymphocytes, a wide range of antigen-specific cells, and many others (all of which can be genetically modified to improve their function, in vivo persistence, or safety). Cells can be expanded in the presence of feeder cells or antigen-presenting cells, including PBMC, PHAblast, OKT3 T, B blast, LCL, K562 (natural or engineered to express antigens, epitopes, and costimulatory molecules such as 41BBL, OX40L, CD80, CD86, HLA, and many others), which may or may not be pulsed with peptides or related antigens.

[0070] Unprecedented Low Initial Surface Density: One aspect of the present invention is the discovery that production time can be reduced relative to conventional methods by using a low cell surface density. In this way, the desired cells can be grown at a greater numerical difference between the minimum and maximum surface density than conventional methods allow. When the growth rate of the desired cell population begins to decline but the quantity of desired cells is still insufficient to terminate production, it is preferable to redistribute the desired cells at a lower surface density onto another growth surface composed of a gas-permeable material.

[0071] To illustrate how our novel cell production method, which relies on a lower surface density at the beginning of any given culture stage, can be applied, an example is now provided. Figure 11 graphically illustrates the population growth of a desired cell on a growth surface under a conventional scenario, compared to the population growth of a desired cell type using one aspect of this invention. In this novel method, the surface density of the desired cells at the beginning of the production stage is lower than the conventional surface density. To focus on the advantages of this novel method, this description does not describe the process of initially obtaining the desired cell population. The "day" of culture begins at "0" so that one skilled in the art can easily understand the relative advantages of this novel method over time. In this example, each production cycle of the conventional method produces 0.5 x 10 desired cells compared to the conventional surface density. 6 pieces / cm 2 While starting at 0.125 × 10, each fabrication cycle in this example reduces the surface density of the desired cells to a much lower, unconventional 0.125 × 10. 6 pieces / cm 2 Thus, in this example, four times the area required to initiate the culture (i.e., 500,000 / 125,000) is required for the conventional method. In this example, the desired cells for the conventional method reach a maximum surface density of 2 x 10 cells in 14 days. 6 pieces / cm 2 Therefore, 1 cm 2 The growth area of ​​2 × 10 cells 6 pieces / cm 2 Then, 0.5 × 10 conventional cells were added. 6 pieces / cm 2 (i.e. 4 cm 2 x0.5 x 10 6 pieces=2×10 6 This is then increased to 4 cm so that fabrication can continue with a starting density of 100 pieces. 2 This cycle is repeated for another 14 days, at which point maximum cell surface density is again reached and the cells are redistributed over a growth area of ​​4 cm. 2 Each of the growth areas is 2 x 10 6 cells, a total of 8.0 × 10 6 This is then divided into 16 cm2 This growth cycle was repeated to obtain a total of 32 x 10 6 Generate cells.

[0072] The novel method depicted in Figure 11 uses a 1 cm 2 Instead of using the conventional method of placing 500,000 target cells on a 4 cm 2 500,000 cells were evenly distributed over the growth area of ​​125,000 cells / cm on day 0. 2 In this example, the new method, like the conventional method, begins to decrease in growth rate at day 7. The new method produces cells at an initial surface density of 1 x 10 6 pieces / cm 2 Therefore, at the point where the growth rate begins to decrease, this stage of the culture is at a surface density of 4 × 10 6 The cells are then cultured in a 32 cm 2 The second step was to produce 0.125 x 10 cells by redistributing the cells over the growth area of ​​0.125 x 10 cells. 6 pieces / cm 2 (i.e. 32 cm 2 x 0.125 x 10 6 pieces=4×10 6 The cell surface density can continue to be increased to a starting surface density of 32 cm. The production cycle or steps are repeated for an additional 7 days, up to day 14, at which point the cell surface density again reaches a maximum of 32 cm. 2 1.0 x 10 for each unit area of ​​growth surface 6 The desired cells were produced, totaling 32 × 10 cells in just 14 days. 6 cells are produced. Notice how, at the end of each production cycle, this new method produces a factor of final surface density divided by the starting surface density, just as in conventional methods. However, by lowering the starting surface density and completing each stage of production before the cells enter growth, production time is dramatically reduced. This example shows how the surface density of the desired cells is reduced relative to the conventional cell surface density (in this case 0.125 x 10 cells). 6 pieces / cm 2) can generate the same amount of desired cells as traditional methods in just 33% of the time (14 days vs. 42 days).

[0073] We used 0.125 x 10 cells 6 pieces / cm 2 Those skilled in the art should be aware that, although the benefits were quantified using a starting surface density of 0.01, this example of the invention illustrates that any reduction below conventional surface densities will result in reduced production times. Furthermore, in this and other novel methods provided herein, those skilled in the art will appreciate that the cell growth rates and points at which cell growth slowdown occurs are merely illustrative, and that the actual rates will vary for each application based on various conditions, such as media composition and cell type. Also, while the specific conventional surface density used in this illustrative example for a given application may vary from application to application, those skilled in the art will appreciate that an advantage of this aspect of the invention is the reduced production times realized as a result of reducing cell surface densities below conventional cell surface densities in any given application.

[0074] Thus, one aspect of the method of the invention is described herein when there is a desire to reduce production time by reducing cell surface density for a given amount of cells of interest present in a cell population, as follows: the cells of interest must be placed on a growth surface at an unprecedentedly low cell surface density. a. The cells of interest are in the presence of antigen-presenting cells and / or feeder cells, and the medium volume to surface area ratio is 1 ml / cm if the growth surface is not composed of a gas-permeable material. 2 up to 2 ml / cm if the growth surface is constructed of gas-permeable material 2 Until b. The preferred surface density condition at the start of the production cycle is a target cell surface density of preferably 0.5 x 10 6 pieces / cm 2 and more preferably as depicted in FIG. 4, c. The surface density of the cells of interest plus the surface density of the antigen-presenting cells and / or feeder cells is preferably at least about 1.25 x 10 5 pieces / cm 2 is.

[0075] Based on the example above, if the surface density of antigen-presenting cells and / or feeder cells is 1.25 x 10 cells 5 pieces / cm 2 It would be prudent to demonstrate that the expansion of the desired cell population does not become limiting when attempting to further reduce the density below 1.25 × 10 cells. With the goal of demonstrating that expansion of the desired cell population can be achieved at unprecedentedly low densities when augmented with an appropriate supply of antigen-presenting cells and / or feeder cells, we have demonstrated that the density of 1.25 × 10 cells is 1.25 × 10 cells. 5 pieces / cm 2 was selected.

[0076] The use of growth surfaces constructed from gas permeable materials and large medium volume to growth area ratios simplifies and shortens fabrication. Another aspect of this invention is the discovery that fabrication times can be shortened by using growth surfaces constructed from gas permeable materials, medium volume to growth area ratios that exceed conventional ratios, and repeated fabrication cycles that increase the amount of growth surface over time.

[0077] To show how these conditions reduce fabrication time, an illustrative example is provided here. Figure 12 shows a growth surface constructed from a gas-permeable material and a 1 or 2 ml / cm 2 This supplements the discussion that illustrates examples of the advantages that can be obtained by utilizing unprecedentedly large medium volume to growth surface area ratios, such as those exceeding 1000 rpm. The following discussion is intended to demonstrate to one of skill in the art that several options are available using such methods, including reducing preparation time, the amount of growth surface used, and / or reducing labor and contamination risks. Those skilled in the art will appreciate that Figure 12 and the associated discussion are examples only and are not intended to limit the scope of the invention.

[0078] The cell mass containing the desired cell population in this illustrative example is assumed to consume approximately 1 ml per "X" length of time. Figure 12 shows two production processes, labeled "Traditional Method" and "New Method." At the start of growth, each process consumes 0.5 x 10 6 / cm 2 However, the growth surface in the new method is constructed from a gas-permeable material, and the medium volume to surface area ratio is reduced from 1 ml / cm2, as compared to the conventional method. 2 whereas 2 ml / cm 2 At time "X", the population of cells of interest in the conventional method is at a surface density of 2 x 10 6 / cm 2 The growth plateau is reached and nutrients are depleted. Meanwhile, the additional medium volume of the novel method allows growth to continue, resulting in a surface density of the desired cells of 3 × 10 6 / cm 2 If we continue with the new method, the surface density will be 4×10 6 / cm 2 This creates many valuable options. The new method can stop before time "X" and produce more cells than the conventional method, or it can stop at time "X" and produce approximately 1.5 times more cells than the conventional method, or it can produce twice as many cells in twice the time as the conventional method without having to manipulate the equipment to continuously feed the media nutrients until they are depleted. To collect as many cells as possible with the conventional method, the cells must be harvested and the process started again, adding labor and risk of contamination. Cell therapy applications typically only start with a certain number of cells, so simply increasing the surface area at the start of production is not an option with the conventional method.

[0079] Figure 13 continues the example of Figure 12 and illustrates how multiple production cycles can be even more beneficial. Figure 13 graphically illustrates the growth of a population of cells of interest on a growth surface under conventional methods compared to the growth of a population of a cell type of interest under one of the novel methods of this invention. Here, the surface density of the novel method exceeds the surface density of the conventional method. To focus on this example, this description will not discuss the process of obtaining the desired cell population. The "days" of culture are counted from "0" to allow one of skill in the art to more easily determine the relative time advantages of this aspect of the invention. In this example, both cultures were grown at a density of 0.5 x 10 cells compared to the conventional method. 5 pieces / cm 2 The cells are grown on "Day 0" with a desired cell surface density of 4 ml / cm. In this illustrative example, the growth surface in the conventional method is also comprised of a gas permeable material. However, the medium volume to growth surface ratio in the conventional method is 4 ml / cm in the new method. 2 1 ml / cm 2 As shown in Figure 13, the population of the desired cells in the conventional method reached a cell surface density of about 1.5 x 10 in about 4 days. 6 pieces / cm 2 The growth rate began to slow down at 14 days, reaching a maximum cell surface density of 2 × 10 6 pieces / cm 2 At that point, the population of cells of interest reaches a density of 1.0 ml / cm 2 0.5 x 10 in fresh medium 6 / cm 2 4 cm at a surface density of 2 The production cycle then begins again, with the cell surface density increasing to 2 x 10 over the next 14 days. 6 pieces / cm 2 reached 8 x 10 in 28 days. 6 In comparison, the population of desired cells in the novel method grows to approximately 3 x 10 cells in approximately 10 to 11 days. 6 pieces / cm 2 The growth rate began to slow down at a surface density of 4 × 10 cells, reaching a maximum cell surface density of 4 × 10 cells at 28 days. 6 pieces / cm 2However, to accelerate production, the cycle is terminated when the desired cell population is still at a high growth rate. Thus, in approximately 10 to 11 days, 3 x 10 6 of cells at 4.0 ml / cm 2 in fresh medium at a surface density of 0.5 x 10 6 / cm 2 6 cm 2 The production cycle then begins again, and the desired cell population grows to a cell surface density of 3 x 10 over approximately another 10-11 days. 6 pieces / cm 2 The target cells reached 18 × 10 in approximately 21 days. 6 Therefore, the new method produced more than twice the number of target cells in approximately 75% of the time compared to the conventional method.

[0080] We cultured 10 × 10 cells on a growth surface constructed from gas-permeable material. 6 pieces / cm 2 Cell surface densities in excess of 1000 μg / cm2 were obtained, and the high surface density aspect of our invention is not limited to the densities mentioned in this example.

[0081] Therefore, another example of the method of the invention is described herein when there is a desire to minimize production time by using a small cell surface density for a given amount of cells of interest present in the cell population. a. In the presence of antigen-presenting cells and / or feeder cells, and with a medium volume to surface area ratio of at least 2 ml / cm 2 seeding cells of interest onto a growth surface constructed from a gas-permeable material; b. At the beginning of the production cycle, establish the preferred surface density conditions to achieve a target surface density of approximately 0.5 x 10 cells. 6 pieces / cm 2 Within the conventional density range of c. The population of desired cells is approximately 2 x 10 conventional cells. 6 pieces / cm 2 allowing proliferation beyond the surface density of d. If more target cells are needed, redistribute the target cells onto another growth surface comprised of gas permeable material and repeat steps a through d until sufficient target cells are obtained.

[0082] Further advantages can be achieved using this novel method by combining the features of using an unprecedented low surface area to initiate the culture, using novel surface densities of cells of interest and feeder cells, using growth areas constructed from gas permeable materials, using unprecedented high medium volume to growth area ratios, and performing production in cycles. Conditions can be varied to achieve a desired outcome, such as finding a balance between a given production cycle, reduced time, surface area utilization, feeding frequency, etc.

[0083] Figure 14 illustrates another novel method that provides additional advantages over conventional methods. As with the other illustrative examples described herein, those skilled in the art will recognize that this description is not intended to limit the scope of the invention, but rather to illustrate how the advantages of improved fabrication efficiency may be realized.

[0084] In this example, the cells of interest would double in one week under conventional conditions. The "day" of culture begins on day "0" to allow one skilled in the art to more easily assess the relative time advantages of this example. Also, for simplicity of this example, the issues previously discussed regarding the ratio of feeder cell surface density and / or antigen-presenting cell surface density will not be repeated. For purposes of illustration, assume that a starting population of 500,000 cells of interest, which would double in seven days under conventional conditions, is present on production "day 0." Conventional methods produce 0.5 x 10 cells. 6 pieces / cm 2 and a surface density of 1 ml / cm 2 As shown, the desired cell population is 2 x 10 6 pieces / cm 2 When the cells reached a surface density of 0.5 x 10 cells 6 pieces / cm 2and start the fabrication cycle anew. 6 pieces / cm 2 surface density of 6 ml / cm and a growth area constructed from gas permeable material. 2 As shown, when population growth approaches the beginning of a plateau, cells are redistributed over a larger growth area. In this case, the cell surface density is 1.5 times the medium volume to surface area ratio (i.e., approximately 1.5 × 10 6 When the population approaches 4.5 × 10 cells / ml, the conventional method determines that the plateau region has begun, and the population is considered to have reached a plateau. 6 pieces / cm 2 At a surface density of 36 cm 2 are distributed over the growing area and the production cycle begins anew.

[0085] Figure 15 tabulates a comparison of each of the fabrication methods depicted in Figure 14, spanning multiple stages to demonstrate the power of the new method and why it is prudent to adjust the fabrication design at various stages to fully exploit efficiency. Note that immediately after completing the second stage of the fabrication cycle, the new method outperforms the conventional method, producing 1.37-fold more cells in approximately half the time, at only 61% the surface density requirement. However, note how the third stage of fabrication massively increases the number of cells, with a corresponding increase in surface area. Therefore, to achieve optimal efficiency levels for any given process, it is necessary to model the fabrication cycle to predict how to adjust the initial and / or final cell surface densities throughout each cycle of the process.

[0086] For example, Figure 16 shows how variables can be changed in the novel method to gain efficiency as the fabrication progresses. For example, the initial surface density in cycle 3 can be increased from 0.06 to 0.70 cells / cm. 2 with final surface densities of 4.5 to 7.5 cells / cm2 Increasing the final surface density can be achieved by increasing the initial surface density from 6 ml / cm 2 The idea is to increase the medium volume to surface area ratio beyond 0.1 ml / cm to a larger value. The higher the medium volume to surface area ratio, the longer the cycle will remain in the rapid growth phase (i.e., population growth before plateauing). In this case, we use an extra 5 days to complete the rapid growth phase, increasing the medium volume to surface area ratio to about 8 ml / cm. 2 By doing so, in this example, it is possible to produce over 3 trillion cells in 34 days on a reasonable surface area. For example, we have developed a 625 cm2 substrate constructed from gas-permeable materials. 2 They fabricated and tested a device with a growth surface of 100 μm, which is a clearly superior approach to cell production than conventional methods.

[0087] Therefore, another preferred embodiment of the invention is described herein when there is a desire to minimize production time by reducing cell surface area for a given amount of cells of interest present in a cell population. a. In the presence of antigen-presenting cells and / or feeder cells, and with a medium volume to surface area ratio of at least 2 ml / cm 2 seeding cells of interest onto a growth surface constructed from a gas-permeable material; b. Establish preferred surface density conditions at the beginning of the production cycle to achieve a surface density of the desired cells less than the conventional density, preferably about 0.5×10 6 pieces / cm 2 and approximately 3900 pieces / cm 2 The total number of cells of interest and antigen-presenting cells and / or feeder cells should be at least about 1.25 x 10 5 pieces / cm 2 west, c. The population of desired cells is approximately 2 x 10 6 pieces / cm 2 allowing proliferation beyond the surface density of d. If more target cells are needed, redistribute the target cells onto another growth surface comprised of gas permeable material and repeat steps a through d until sufficient target cells are obtained.

[0088] This invention provides cell culture devices and methods that allow for much better cell generation, particularly for the field of adoptive cell therapy. Various advantages are possible over current devices and methods, including reduced time required to provide a given number of cells, greater expansion of the desired cell population from the initial cell mass, reduced or elimination of frequent medium changes, simplified cytokine addition, reduced or elimination of the need to count cells to determine cell mass, significantly reduced medium volume required to separate cells from a previous culture, and more efficient generation and proportional expansion of antigen-specific cell populations.

[0089] Example 9: A more efficient method of producing cells in static gas permeable culture devices by setting novel culture conditions at the beginning of the culture process.

[0090] Static cell culture experiments were performed by culturing K562 cells in a test fixture constructed with a growth surface made of a gas-permeable silicone material and with a wall height that allowed 10 cm of medium to reside above the growth surface. The growth surface was held in a nearly horizontal position using a growth surface support, as described in more detail in Wilson '717. Medium was placed in the test fixture with a medium height of 10 cm above the growth surface, and a flow rate of 10 ml / cm was used. 2 A medium volume to growth surface ratio of 1000 nm was achieved. K562 cells were also placed in the test device, and the device was placed in a cell culture incubator at 37°C, 5% CO2, and 95% RH, allowing the cells to settle to the growth surface. The medium was not perfused or forcibly agitated, and gas was not forced to flow past the growth surface, but rather was brought into contact with the growth surface by random movement of the ambient atmosphere.

[0091] For illustrative purposes, Figures 17A, 17B, 17C, 17D, and 17E show spreadsheets depicting experimental conditions and typical results. Initial static culture conditions involved cell surface densities ranging from 1.0E+06 to 6.25E+04 cells / cm. 2 The cell density was set to a range of 1.0E+05 to 6.25E+03 cells / ml during the incubation period. The medium was maintained at a constant height of 10 cm above the growth surface in all conditions. All medium had the same composition and glucose concentration of 240 mg / dL. The initial state of the static culture was day 0, and cell counts and glucose concentrations were measured on days 4, 8, and 11.

[0092] Figure 18 compares the fold expansion of population versus surface density for each of the experimental conditions detailed in Figures 17A through 17E. The fold expansion for each condition was calculated by dividing the cell surface density on day 11 by the cell surface density on day 0. The lower limit of surface density was 5.0E+05 cells / cm. 2 In a series of evaluations with a maximum medium height of 2.0 cm, we concluded that the best expansion fold of K562 in gas-permeable bags was approximately 4.8-fold. Therefore, dotted line 6 shows a typical expansion fold for conventional K562 production methods using gas-permeable bags. At each surface density condition established in our experiments, we obtained a population expansion that exceeded that of the conventional technology. Notably, the ability to increase the cell population expansion fold increases as the initial static culture surface density decreases down to 0.125E+06, and further reductions, while still significantly better than conventional methods, offer little advantage.

[0093] Other observations were made to further explore whether glucose could be a surrogate measure of cell number at any time and specifically the ability to run cultures for extended periods without feeding.

[0094] Example 10: A novel method for determining the amount of cells in a population present in a static gas permeable culture device without the need to count the cells.

[0095] We observed that the rate of glucose depletion was a consistent indicator of the number of cells in the culture, even though the culture medium was static and not mechanically forced to mix by perfusion, shaking, or agitation prior to sampling (other than routine instrument manipulation). This discovery opened the door to further simplifications in the field of adoptive cell therapy. For example, counting cells to determine how well a culture is progressing is one of many factors that makes cell preparation for adoptive cell therapy impractical. By using a surrogate measurement in place of cell counting, combined with the invention herein, cell preparation is made even simpler.

[0096] We have discovered that it is possible to use the glucose concentration of a culture as a surrogate measure of a culture's population. For a culture in which cells reside on a growth surface constructed of a given type of gas-permeable material, knowing the minimum total medium volume required for the culture to reach its maximum surface and the total decrease in glucose concentration required for the culture to reach its maximum surface density, we are ready to make a surrogate prediction of the number of cells in the culture's population. Armed with that information, someone initiating a culture (or a stage of a culture) determines the baseline concentration of glucose in the medium, the baseline volume of medium, and constantly monitors the medium volume added to the culture before measuring the glucose concentration at the time of population assessment. The estimated number of cells in the population is a function of the proportionally proportional total decrease in glucose concentration required to reach the maximum cell density multiplied by the proportionally proportional minimum medium volume required to reach the maximum surface density multiplied by the maximum surface density possible on the growth surface.

[0097] We applied this method to the cultures described throughout the various disclosures of this invention, in which the growth surface of the labware was composed of dimethyl silicone approximately 0.006 to 0.0012 inches thick. A series of experiments was performed to determine the minimum medium volume required for the cells to reach maximum surface density and the corresponding total reduction in glucose concentration. For a variety of cultures with different cell types, including K562, LCL, and T cells, the total reduction in glucose concentration was approximately 250 mg / ml. We were able to develop a relationship to predict the number of cells in culture, as shown below. A = baseline concentration of glucose in the medium B = measured glucose concentration at the time of population estimation C = total decrease in glucose concentration required to reach maximum surface density D = baseline volume of medium E = volume of medium added after baseline F = minimum total medium volume required to reach maximum surface density G=maximum surface density E = area of ​​growing surface Then, [(AB) / C] x [(D+E) / F] x G x E = estimated number of cells in the culture population in the device. Note that the minimum prorated medium volume cannot exceed 100% because adding medium will not increase the surface density above the maximum volume. For example, if a culture requires 10 ml to reach maximum surface density, and the sum of the baseline volume of medium and the volume of medium added exceeds 10 ml, 100% should be used as the minimum prorated medium volume.

[0098] This prediction equation requires that the maximum cell density (and / or maximum surface density) for cell culture applications be known, provided the cells reside on a growth surface constructed from the gas permeable material of the engineer's choice. Experiments can be performed to make this determination. For example, to determine the maximum cell surface density of K562 cells on a growth surface constructed from a gas permeable material (dimethyl silicone, as mentioned above) in our experimental fixture, we increased the medium height until the surface density could no longer be increased. We estimated the maximum achievable surface density of K562 to be approximately 12.0E+06 cells / cm. 2 It was found that the minimum medium volume required to maintain the growth rate was 10 ml, and the corresponding total reduction in glucose concentration was 250 mg / ml.

[0099] An illustrative example of how this information can be used to determine cell number in a K562 culture follows. In the first example, assume that no additional medium is added after the culture is initiated and that the following conditions exist: Baseline volume of medium = 10 ml Baseline glucose level = 475 mg / dl Medium addition = 0 ml Glucose sample = 300 mg / dl Area of ​​growing surface = 100 cm 2 In this case, the calculation would be as follows: [((475 mg / dl-300 mg / dl) / 250 mg / dl)×(10 ml+0 ml) / 10 ml]×12E+0 6 pieces / cm 2 ×100 cm 2 = 840×10 6 pieces.

[0100] As another example, assume that medium is added after the start of the culture and the following conditions exist: Baseline volume of medium = 6 ml Baseline glucose level = 475 mg / dl Addition of medium = 2 ml Glucose sample: 300 mg / dl Area of ​​growing surface = 100 cm 2 In this case, the calculation would be as follows: [((475 mg / dl-300 mg / dl) / 250 mg / dl)×(6 ml+2 ml) / 10 ml]×12E+06 pieces / cm 2 ×100 cm 2 = 672×10 6 pieces.

[0101] As yet another example, assume that medium is added after the start of the culture and the following conditions exist: Baseline volume of medium = 6 ml Baseline glucose level = 475 mg / dl Addition of medium = 7 ml Glucose sample: 300 mg / dl Area of ​​growing surface = 100 cm 2 In this case, the total medium volume added to the culture exceeds the minimum total medium volume required to reach maximum surface density, so the minimum prorated medium volume would amount to 100%, and therefore the prorated value would equal 1, and the calculation would be: [(475 mg / dl-300 mg / dl) / 250 mg / dl×(1)]×12E+06 pieces / cm 2 ×100 cm 2 =840×10 6 pieces.

[0102] The maximum cell density (cells / cm) in culture medium that a particular cell type can achieve when present on a growth surface constructed of a particular type of gas permeable material. 2Those skilled in the art should be aware that another relationship can be used to estimate the number of cells in culture by predetermining the glucose concentration required to reach the maximum cell density. In that case, the relationship is a function of (pro-rata total decrease in glucose concentration required to reach the maximum cell density) x (the sum of the volume of medium with which the culture is started and the volume of medium added to the culture) x (maximum cell density). If the cumulative volume of medium exceeds that of the minimum medium volume required to reach the maximum surface density, it is recommended to use the minimum medium volume instead of the cumulative volume (because the extra medium volume will not increase the surface density beyond its maximum).

[0103] To help understand the predictive power of this equation, we included a prediction of the number of cells in culture in row 10 of each spreadsheet (normalized by growth area) for the conditions shown in Figures 17A through 17E. A comparison of row 10 with the counted cells in row 12 shows how the number of cells in culture at any given time can be determined with a reasonable degree of certainty using glucose instead of cell count. In practice, cell counts may be inaccurate because it is not possible to ensure that the cells are uniformly mixed into the medium before counting. Therefore, it is more useful to rely on glucose measurements. In one preferred embodiment, cell counts are not performed for at least 4 days, more preferably 5 days, more preferably 6 days, more preferably 7 days, more preferably 8 days, and even more preferably until the end of the culture.

[0104] Further experiments were performed to determine whether the formula describing the relationship between glucose depletion and the number of viable cells in the fixture was accurate when the glucose concentration at the start of the culture was varied. The test fixture was the same as described above. For illustration, Figure 19A shows a spreadsheet depicting the experimental conditions and typical results for the culture of K562 cells under the same starting conditions, except for the glucose concentrations at the start of the culture, which were 240 mg / dL and 475 mg / dL. The results are depicted graphically in Figures 19B, 19C, 19D, 19E, and 19F. Population growth by cell count and that predicted by glucose depletion were normalized by surface density.

[0105] Figure 19B shows population growth over 11 days under each condition. While population growth varied slightly, they reached roughly the same populations at 11 days. Figure 19C shows the rate of glucose decline for each culture condition. Figure 19D shows glucose consumption for each culture condition. Figure 19E shows the predicted cell number formula along with the cell number determined by manual counting for cultures initiated at a glucose concentration of 240 mg / dL. Figure 19F shows the predicted cell number formula along with the cell number determined by manual counting for cultures initiated at a glucose concentration of 475 mg / dL. Note that the predictive ability of the formula is close to that of the manual cell counting method. This further demonstrates that various embodiments of the present invention can be used in conjunction with methods that reduce or eliminate cell counting frequency in lieu of alternative measures of solute concentration in the medium.

[0106] This is a particularly strong advantage over cell counting when one wishes to use the gas-permeable devices described in Wilson '717 or Wilson '176. Those skilled in the art will recognize the difficulty of accurately distributing cells in such devices and the potential for miscounting due to poor cell distribution in the medium. Therefore, a surrogate measurement that relies solely on medium samples instead of actual cell counts would be highly advantageous in the field of adoptive cell therapy.

[0107] Armed with this knowledge, manufacturers of gas permeable devices, including those described in Wilson '717 or Wilson '176, can provide a simplified cell culture device having a growth surface constructed from a gas permeable material, and by providing instructions and / or disseminating information regarding this invention disclosure, provide a simplified cell culture process in which the number of viable cells within the gas permeable device can be easily determined without the need for cell counting.

[0108] Example 11: A simpler method for producing cells in static gas permeable culture devices by establishing new conditions at the beginning of the culture process to limit feeding frequency in static cultures.

[0109] We conducted a series of experiments to determine the ability to reduce the frequency of feeding by using the novel method disclosed herein compared to conventional culture methods that require feeding every two or three days.

[0110] Experiments were conducted with varying medium height capacities in devices with growth surfaces constructed from gas-permeable materials. The following description of an experiment comparing medium volume and feeding frequency explains our findings. K562 cells and medium were placed in the devices and placed in a cell culture incubator at 37°C, 5% CO2, and 95% RH. Doing so resulted in a growth rate of 0.125E+06 cells / cm, which was found to be advantageous for achieving a superior population expansion ratio, as previously described. 2 At a surface density of 1000, the cells were allowed to settle onto the growth surface.

[0111] Silicone area 100 cm 2 The medium was placed at heights of 2.5 cm, 5.0 cm, 10.0 cm, and 15.0 cm above the growth surface. The growth surface was held in a nearly horizontal position using a growth surface support, as described in detail in Wilson '717. Therefore, the experimental conditions included a medium volume to growth surface area ratio of 2.5 ml / cm. 2 , 5.0 ml / cm 2 , 10.0 ml / cm 2 , and 15.0 ml / cm 2 Therefore, the initial cell densities were 0.05E+06 cells / ml, 0.025E+06 cells / ml, 0.0125E+06 cells / ml, and 0.008E+06 cells / ml, respectively.

[0112] 10.0 ml / cm 2 or 15.0 ml / cm 2 No further medium was added under the condition of 2.5 ml / cm 2 The initial medium volume of the condition was 2.5 ml / cm on day 11. 2 Add fresh medium to double the number of cells and on day 14 add another 2.5ml / cm 2 Adding fresh medium tripled the number of cells and then adding another 2.5 ml / cm on day 17. 2 Adding fresh medium at 5.0 ml / cm increased the number of cells by 4 times. 2 The initial medium volume of the condition was 5.0 ml / cm on day 11. 2 The number of cells doubled with the addition of fresh medium. Finally, the number of cells was increased to 2.5 ml / cm. 2 and 5.0 ml / cm 2 The condition is 10.0 ml / cm 2 The medium was maintained at 100°C.

[0113] FIG. 20 shows a graph of population growth normalized by growth area under various media feeding conditions.

[0114] Note that all conditions ultimately reached roughly the same number of viable cells. However, the condition that did not rely on additional medium during culture reached the maximum number of viable cells more quickly than the other conditions. For example, 2.5 ml / cm 2 It took 20 days for the 15.0 cm condition to reach maximum density, while it took only 11 days for the 10.0 cm condition in which fresh medium was not added after culture initiation to reach the same maximum number of viable cells. Furthermore, the population growth rate was much better in the unfed condition. Importantly, the 15.0 cm condition demonstrated the ability of cells to maintain high viability for a longer period of time compared to the 10.0 cm condition. For example, viability was relatively high for approximately 4 days after reaching maximum cell population in the 15.0 cm condition, whereas it rapidly declined after approximately 1 or 2 days in the 10.0 cm condition. A practical benefit accrues here: the longer cell harvesting process. Those skilled in the art of adoptive cell therapy will appreciate this, as there are many reasons why one might find value in harvesting cells over a longer time frame, ranging from delays in obtaining quality control measurements to changes in patient status.

[0115] Those skilled in the art will recognize that all of the experimental culture conditions have superior cell population expansion rates compared to conventional methods for adoptive cell therapy. However, it should be noted that not only does it have the advantage of lowering the surface density at the start of the culture compared to conventional methods, but it can also shorten the time required to generate the desired number of cells by increasing the medium height and / or the medium-to-growth surface area ratio. Those skilled in the art should recognize that lowering the surface density, increasing the medium height, and / or increasing the medium volume-to-growth surface area ratio will also result in improvements in population expansion rate, and are encouraged to balance medium usage with the needs of the application. If a longer window is desired for harvesting cells while they remain highly viable, more medium can be provided at the start of the culture. It is noteworthy that even when starting at conventional or even higher surface densities, such as 2.0E+06 or higher, embodiments of the present invention are superior because they allow for less frequent feeding and reduce the risk of the cell population rapidly losing viability. In general, a wide range of options has been presented. To generate a cell population with minimal feeding frequency and reduced production time, the most favorable initial culture conditions for production are 0.5E+06 cells / cm 2 A cell density of less than or equal to 0.125E+06 cells / cm is most preferred. 2 and a medium height of about 5.0 cm or more, or more preferably 10.0 cm to 15.0 cm, and / or about 5.0 ml / cm 2 or more preferably 10.0 ml / cm 2 to 15.0 ml / cm 2 and / or 0.025E+06 cells / cm 2 or less, or more preferably about 0.0125E+06 particles / cm 2 Approximately 0.008E+06 pieces / cm 2 is the initial cell surface density.

[0116] Example 12: A novel method for reducing the feeding frequency of a co-culture present in a static gas permeable culture device and determining the cell population size without the need to count cells even when some of the cells are dying.

[0117] Adoptive cell therapy typically relies on co-cultivation with cells that are dying due to irradiation (e.g., APCs) or cells that are dying as a result of being removed from the human body (e.g., PBMCs). A good example of a co-cultivation application is the cultivation of CMV-CTLs (cytomegalovirus-specific cytotoxic T lymphocytes) from a population of PBMCs. Initially, the CMV-CTL population is a very small percentage of the total PBMC population. As the culture progresses, PBMCs begin to die and CMV-CTLs begin to grow. By the end of the culture, the proportion of CMV-CTLs in the cell population has greatly increased. The previously disclosed properties of this invention, including those contrary to conventional methods, such as low cell density, can be used to reduce the frequency of feeding for applications such as these.

[0118] Static cell culture experiments were performed to examine the ability of glucose measurements to predict cell population in the presence of dying cells in co-culture. 2 The growth surface was constructed of gas-permeable silicone with a surface area of ​​1.5E+0.5 cells / cm. The growth surface was constructed of silicone and held in a generally horizontal position using a growth surface support as described in more detail in Wilson '717. Figure 21 shows a spreadsheet summarizing the conditions on days 0, 9, and 16. PBMC medium was placed into the labware, and the labware was placed in a cell culture incubator at 37°C, 5% CO2, and 95% RH. This allowed for a growth of 5.0E+0.5 cells / cm. 2The cells were allowed to settle above the growth surface at a surface density of 0.0E+0. The medium was present at a height of 10.0 cm, and the cell density was 5.0E+0. Glucose measurements were taken on days 0, 9, and 16. Except for routine manipulations, the culture medium and cells were not mixed by forced mixing with the aid of mechanical devices, such as perfusion, shaking, or stirring. Row 3 shows that the proportion of antigen-specific T cells increases to approximately 27.9% of the population, and row 4 shows how the fold expansion of CMV-CTL declines as a proportion of the total population from day 9 onwards, as PBMCs begin to die.

[0119] Line 19 shows the ability of surrogate measurements of solutes in the medium to predict the number of cells in culture. Note that the predicted values ​​are nearly identical to the cells examined by counting. Thus, the ability to use surrogate measurements to quantify cell populations is useful even in cell populations where components of the cell population are dying.

[0120] Example 13: A novel static gas permeable cell culture and cell harvesting device that allows for a simplified method of medium exchange, and a novel method that greatly reduces the effort required to separate cells from the medium after completing the cell production process.

[0121] FIG. 22A shows a cross-sectional view of one example of an embodiment of a cell culture and cell harvesting device 1000 configured to perform the novel cell culture and / or cell harvesting methods disclosed herein. The cell removal opening 1002 of the cell removal tube 1004 is located near the growth surface 1006. The medium removal opening 1008 of the medium removal tube 1010 is located near the growth surface 1006. The growth surface 1006 is comprised of a gas-permeable material. Numerous sources exist for those skilled in the art to learn about suitable gas-permeable materials, including Wilson '717. Preferably, the growth surface 1006 is liquid-tight and non-porous. The distance from the growth surface 1006 to the upper limit 1012 of the interior volume 1004 determines the volume of space available for medium to reside. Although medium can be present in the medium removal tube 1010 and cell removal tube 1004 that extend to a height greater than the upper limit 1012, those skilled in the art should consider the maximum medium height to be the maximum distance from the bottom of the internal volume 1014 to the upper limit 1012 for purposes of describing this embodiment. This cell culture and cell harvesting apparatus does not require a stirring mechanism or any other mechanism to mix the cells and / or medium.

[0122] FIG. 22B shows the cell culture and cell collection device 1000 in an initial static culture state at the beginning of any given cell production stage of the culture. The cell culture and cell collection device 1000 is oriented with the growth surface 1006 oriented horizontally, and cells 1016 have settled on the growth surface 1006. In this illustrative embodiment, a growth surface support 1018 is used to hold the growth surface 1006 in a horizontal position while allowing ambient gas to contact the growth surface 1006 without the need for a pump or other mechanism to flow gas to the growth surface 1006. Those skilled in the art can refer to Wilson '717 for information on how to configure the growth surface support 1018. While the culture medium 1020 can reside at any level within the boundaries of the interior volume 1014, it is preferred that the top medium position 1022 be parallel to the growth surface 1006 as shown. The cell culture and cell collection device 1000 is placed in an atmosphere suitable for cell culture and at a temperature suitable for cell culture. The atmospheric gas contacts the gas permeable material of the growth surface 1006 by random movement without the need for a pump or other mechanism to move the gas to or from the growth surface 1006.

[0123] The height of the medium is determined by the distance from the bottom of the medium to the top of the medium; in this case, the distance from the growth surface 1006 to the top of the medium at the start of the culture is the medium height of the initial static culture. The ratio of the number of cells 1016 that have settled onto the growth surface 1006 to the volume of the medium 1020 is the cell density of the initial static culture. The ratio of the number of cells 1016 on the growth surface 1006 to the surface area of ​​the growth surface 1006 is the surface density of the initial static culture. The ratio of the volume of the medium 1020 to the surface area of ​​the growth surface 1006 is the medium volume-to-growth surface ratio of the initial static culture. The cells exist in a static culture state, and the culture continues for a certain period of time. As discussed throughout this specification, this time may or may not include a medium replenishment step, depending on variables including initial static culture medium height, initial static culture cell density, initial static culture surface density, and / or initial static culture medium volume to growth surface ratio.

[0124] FIG. 22C illustrates another step in harvesting cells from the cell culture and cell harvesting device 1000 in a smaller medium volume. Cells 1016 are not extracted, but the medium is removed through the medium removal opening 1008 of the medium removal tube 1010. The medium remaining after this step is illustrated as cell recovery medium 1024. The less cell recovery medium remaining, the less laborious the process of separating cells from the medium, which is essential in traditional methods for adoptive cell therapy and currently relies heavily on centrifugation. However, it is important to be careful not to lose too many cells when reducing the medium volume. Preferably, no more than 10% of the cells are lost, and more preferably, substantially no cells are lost. To provide further guidance, we provide an example using this aspect of the invention. 100 cm 2 The growth surface is constructed of gas permeable silicone with a growth area of ​​200 ml / cm. The medium volume is 2000 ml, and the height when the medium volume is reduced is 20 cm, thereby achieving a growth rate of 200 ml / cm. 2 In a gas-permeable device similar to that shown in Figure 22A, where the medium volume to growth surface ratio is 0.25, and where a cultured cell population of approximately 1 trillion cells resides above the growth surface upon medium volume reduction, we have demonstrated the ability to avoid visible cell loss while achieving a 100-fold reduction in medium volume and a cell collection medium height of only 0.2 cm (measured when the growth surface was in a horizontal position) and a cell recovery medium rate of only 0.2 ml / cm. 2We established a set of conditions at the time of cell collection, characterized by the ratio of cell collection medium volume to growth surface area. Next, we vortexed the medium, in this case the medium in the device, which instantly lifted the cells from the growth surface and distributed them throughout the cell collection medium. Next, we removed the cells through the cell collection opening of the cell collection tube. The cell collection opening was located along the edge of the device, and we tilted the device so that the medium collected at the cell collection opening. Immediately after collecting the cells and cell collection medium, we examined the cell concentration in the cell collection medium. Surprisingly, it was approximately 50 million cells per ml. Therefore, we were able to concentrate the cells by a factor of 100 from an initial cell density of approximately 500,000 cells per ml without using a centrifuge, as is traditionally used in static cell culture for adoptive cell therapy. As a result, the additional culture process required for cell collection was minimal. Essentially, we reduced the medium volume required for centrifugation from 2,000 ml to only 20 ml, and the entire process took less than one minute.

[0125] The medium removal opening of the medium removal tube is preferably located at a distance of 0.2 cm or more from the growth surface when the growth surface is in a horizontal position. For example, a distance between 0.2 cm and 2.0 cm from the growth surface when the growth surface is in a horizontal position allows for significant volume reduction in many of the cell culture methods of this invention. The upper limit of the distance between the medium removal opening and the growth surface when the growth surface is in a horizontal position is preferably a distance that takes into account the typical height of the medium at the time the medium is reduced for cell harvest. For example, if one were to reduce the volume of medium that needs to be centrifuged by 50% compared to conventional methods for static cell culture, the medium removal opening of the medium removal tube would be inserted to 50% of the medium height (assuming the device is designed to allow medium to reside across the entire growth surface). Due to the premium for using laboratory space, the height of the device should be approximately the same as the height of the medium expected to reside therein. Therefore, to provide the option of obtaining at least a two-fold reduction in medium volume processing over conventional methods, a good guideline is to design the device so that its height is exactly the same as or slightly higher than the typical medium height during use, and to position the medium removal opening of the medium removal tube from approximately 0.2 cm above the growth surface (when the growth surface is horizontal) to approximately halfway from the top of the device to the growth surface, measured from the inside of the device. For example, if the distance from the upper boundary of the growth medium in the device to the growth surface represents the possible height of the medium in the device, when the growth surface is horizontal and at 50% or less of the possible medium height, the medium removal opening should preferably be located 0.2 cm or more above the growth surface. When there is uncertainty about the medium height, multiple medium removal tubes can be provided within the device.

[0126] Preferably, the cell removal opening of the cell removal tube is located along the bottom edge of the device so that the cell collection medium can be collected without reorienting the device. However, the device can be reoriented if desired. Figure 22D illustrates the process of reorienting the cell culture and cell collection device 1000 from its original horizontal cell culture position to a position at an angle 1026, which repositions the cell collection medium 1024 with cells 1016 distributed therein relative to the cell removal opening 1002 of the cell removal tube 1004, allowing the cell collection medium 1024 to be subsequently withdrawn.

[0127] However, the cell removal opening of the cell removal tube need not be located along the bottom edge of the device. After the step of mixing the cells in the cell recovery medium is complete, the cell recovery medium can be removed from any location within the device by simply rotating the device until the cell recovery medium is positioned at the cell removal opening and then removing the cell recovery medium from the cell removal tube. Those skilled in the art will appreciate that the tubes do not have to be as shown, but can have any configuration. A key feature of the design is the ability to position the medium removal opening in a desired location relative to the growth surface, as described above. Thus, the tubes can be as simple as a bulkhead on the side of the device or as complex as nested tubes. Those skilled in the art should also recognize that cell culture and cell recovery methods need not rely on closed system structures, but can be accomplished by simple means such as open system structures. For example, we were able to achieve the above-described concentration by repeating the above-described method and steps even using an open system device of the type described in Wilson '717 and using pipettes as the medium removal tube and cell removal tube.

[0128] To take advantage of the large cell culture medium volume to growth surface area ratio described in various embodiments of this invention, the internal height of the cell culture and cell collection device is preferably at least 2.0 cm or greater for any particular application. Also, to facilitate cell culture and cell collection, the cell culture and cell collection device is preferably constructed of biocompatible materials, transparent for visual evaluation, robust, and easy to handle.

[0129] The discovery of a method for removing medium from the cell culture and cell collection device of this invention without removing cells provides another advantage over conventional static cell culture methods related to the medium change process in adoptive cell therapy. While this specification describes a novel method that avoids medium removal and medium replacement to replenish the medium, there may be situations in which a technician would want to implement this process. Conventional methods result in the removal of cells when removing or replacing medium, and therefore, a common practice is to remove the medium, distribute it to one or more new devices, and then add medium to all of those devices. This results in more devices being used each time a feed is performed. This is not necessary with our novel method, because our cell collection method of this invention leaves the cells in the device when removing the medium using a tube that can be as simple as a pipette. There is no need to use any screens, filters, or centrifugation of the device to reduce the medium volume without cell loss. The medium is simply removed and added to the same device until the cell population reaches maximum surface density.

[0130] In the case of medium removal or replacement, it has already been described how medium can be removed to a height of only 0.2 cm without loss of cells, so it is now easy to understand that medium replacement can be performed without removing any cells by simply removing medium to the desired height and / or volume through the medium removal opening of the medium removal tube, and then adding medium to any desired volume or height.

[0131] Thus, using this specification, one skilled in the art should be able to formulate a preferred embodiment of a static gas permeable cell culture and cell collection device comprising: a. a growth surface formed from a gas permeable material; b. The growing surface is horizontal while the device is in operation. c. a medium removal tube having a medium removal opening; d. a cell removal tube having a cell removal opening; e. internal volume and f. an upper boundary that forms a boundary of the uppermost position of the internal volume; g. The distance from the upper boundary to the growing surface is within the allowable medium height, and the distance from the upper boundary to the growing surface is greater than 2.0 cm; h. When the growth surface is in a horizontal position, the distance the medium removal tube is above the growth surface is at least 0.2 cm above the growth surface and does not exceed 50% of the available medium height.

[0132] Preferably, the device comprises relevant portions of the device described in Wilson '717. Also, armed with this knowledge, manufacturers of gas permeable devices can facilitate more efficient methods of cell culture by providing users with cell culture and cell harvesting devices, including those described in Wilson '717, having growth surfaces, medium removal tubes, medium removal openings, cell removal tubes, and cell removal openings constructed from gas permeable materials, and by providing instructions and disseminating information on: a. Adding cells and media into the device; b. The cells are mammalian and of a non-adherent cell type; c. placing the growth surface in a horizontal orientation, the cells on the growth surface, and the cell culture and cell harvesting equipment in an atmosphere suitable for cell culture at a temperature suitable for cell culture; d. allowing the cells to settle onto a growth surface; e. The ratio of the number of cells that settle onto the growth surface to the medium volume is the initial static culture cell density; f. The ratio of the number of cells that settle onto the growth surface to the surface area of ​​the growth surface is the initial static culture surface density; g. The ratio of the medium volume to the growth surface area is the ratio of the initial static culture medium volume to the growth surface area; h. The distance from the lowest to the highest medium level is the initial static culture medium height; i. allowing the cells to exist in static culture for a period of time, and further comprising the steps of: harvesting the cells from the cell culture and cell harvesting device; j. a pre-harvesting step comprising removing a portion of the medium through the medium removal opening of the cell removal tube without extracting cells, wherein the medium volume remaining in the device is the cell recovery medium volume; k. the height of the cell collection medium is the distance from the top of the cell collection medium to the bottom of the cell collection medium when the growth surface is horizontal; l. The ratio of the volume of the cell collection medium to the surface area of ​​the growth surface is the ratio of the volume of the cell collection medium to the growth surface; m. The ratio of the cell collection medium volume to the medium volume is a reduction rate of the medium, and the cell collection step comprises: n. Mixing the cells in a cell collection medium; o. The ratio of the number of cells in the harvest medium to the volume of the cell harvest medium is the harvested cell density; p. Removing the cells and cell collection medium from the cell culture and cell collection instrument through the cell removal opening of the cell removal tube.

[0133] The ratio of cell collection medium volume to growth surface area is at least 0.2 ml / cm 2Preferably, the medium reduction is at least 50%. Those skilled in the art are advised that this method can be utilized with any embodiment of the present invention, including any desired initial static culture cell density, initial static culture surface density, initial static culture medium volume to growth surface area ratio, and / or initial static culture height, that provides the benefits described herein. Those skilled in the art will also recognize that this method includes use with pancreatic islets.

[0134] Example 14: A novel method using static gas-permeable culture for superior generation of CAR T cells.

[0135] The experiment was carried out in a laboratory device with a growth surface having a surface area made of gas-permeable material and variable medium height. 2 The growth surface was constructed of silicone having a surface area of ​​1000 .mu.m and was held in a generally horizontal position using a growth surface support as described in detail in Wilson '717.

[0136] Three conditions were evaluated for the proliferation of transduced antigen-specific T cells (CAR T cells): Evaluation A included CAR T cells in the presence of K562 APC cells, with a media height of 10 cm; Evaluation B included CAR T cells without K562 APC cells, with a media height of 10 cm; and Evaluation C included CAR T cells cultured according to conventional methods.

[0137] Figure 23A shows the conditions for assessment A at the start of the culture and as the culture progressed. Note that at the start of the culture, the ratio of APC cells to CAR T cells was 2:1 and the culture medium was 10 cm high. Figure 23B shows the conditions for assessment B at the start of the culture and as the culture progressed. Note that at the start of the culture, there were no APCs and the culture medium was 10 cm high. Figure 23C shows the conditions for assessment C at the start of the culture and as the culture progressed. Note that at the start of the culture, the ratio of APC cells to CAR T cells was 2:1 and the culture medium was 2 cm high.

[0138] Unlike conventional culture in the field of adoptive cell culture, cytokine stimulation was performed at the time of medium change by adding cytokines (such as IL2) to fresh cells. Thus, cytokine stimulation was performed simultaneously with medium change. However, as described in various examples of this invention, the frequency of feeding was significantly reduced or even eliminated. Therefore, we also used Condition A and Condition B to evaluate the ability to add cytokines without medium change. Instead of medium change, we simply added boluses of IL2 at the same frequency and to the same volume per ml of medium as in the conventional method, without subjecting the medium to any kind of forced mixing to distribute the IL2 throughout the medium.

[0139] Figure 23D shows the total viable cells at various time points during the culture. As can be seen, the number of total viable cells in condition A was far superior to either of the other conditions. Figures 23E1-23E3 show the percentage of CAR T cells at the beginning and end of the culture. Histogram A represents condition A, histogram B represents condition B, and histogram C represents condition C. Condition B demonstrates the drawback of not providing APCs to the culture at the beginning of the culture. When APCs were provided at the beginning of the culture, CAR expression improved from approximately 40% at the beginning to approximately 80% at the end of the culture. Figure 23F shows the total fold expansion of CAR T cells during the culture. It is clear that condition A results in a significantly greater fold expansion than the conventional method shown in condition C.

[0140] Note also that the viable cell population estimates in Assessment A represent the cell population determined by manual counting, as shown in Figure 23G. Furthermore, the devices used for Assessments A and B were capable of withdrawing media from 1000 ml down to 20 ml at the end of incubation using the methods described above without losing any cells.

[0141] Another important finding concerns the presence of APCs in the culture. Clearly, T cells recovered from condition A, due to their higher T cell yield, are more capable of killing tumor cells expressing the relevant antigen (PSCA), resulting in a larger proportion of CAR-expressing T cells (CAR-PSCA) at the end of the culture relative to the starting state. In comparison, due to the lack of APCs at the start of the culture, condition B fails to increase the proportion of CAR-expressing T cells (CAR-PSCA) in the cell population at all over the culture period.

[0142] Figure 23H shows the ability of cells obtained from condition A and condition B to kill tumor cells expressing PSCA and avoid killing cells that do not express the PSCA antigen. The ratio of effector cells to PSCA antigen-expressing cells (DU145 and Capan1) or non-PSCA antigen-expressing cells (293T) was 40:1. Effector cells (i.e., CAR T cells) were obtained from the cultures of condition A and condition B on day 11 of culture.

[0143] Figures 24A, 24B, 24C, and 24D summarize a side-by-side comparison of the population expansion of CAR T cells specific for PSCA and Muc1 using the initial culture conditions described for condition A (except that the antigen expression of the APCs was PSCA and Muc1, respectively). It can be seen that the novel initial conditions of the present invention allow for the generation of much higher numbers of CAR T cells in a shorter time than conventional conditions in conventional culture vessels. Those skilled in the art will appreciate that these advantages are not limited to CAR T cells that recognize the PSCA or Muc1 antigen, but can be applied to CAR T cells that recognize any antigen.

[0144] The ability to generate more cells in a shorter period of time, the ability to add cytokines without the need for medium changes or forced mixing of the medium, the ability to eliminate the need to supply fresh medium to the culture, the ability to avoid the need for manual cell counting, and the ability to reduce the amount of medium in which the cells reside at the time of cell harvest to only 2% of the volume present at the end of the culture demonstrated the power of this invention to overcome many of the problems inherent in conventional methods for generating cells for adoptive cell therapy.

[0145] Example 15: The method of the present invention scales linearly with the area of ​​the growing surface.

[0146] We conducted experiments in the context of the parent application to assess the scalability of the culture process described here and in the parent application. The ability to move from small to large growth areas without having to re-establish experimental designs to optimize the culture process is a powerful advantage. To determine whether this is the case, we cultured 10 cm2 areas of gas-permeable silicone. 2 , 100 cm 2 , 640 cm 2 The results of K562 cultures initiated on a growth surface of 0.125E+06 cells / cm were compared. 2 The surface density was 100 μm and the medium height was 10 cm. After the culture was initiated, the medium was not replenished.

[0147] Figure 24E shows population growth curves for three gas-permeable culture devices with different growth areas. Series 1 was 640 cm 2 Series 2 represents the viable cell growth of cultures in the device, 100 cm 2 The instrument, and the Series 3, 10 cm 2 Figure 24F shows the population growth curves of Figure 24E normalized by surface density, demonstrating clear linear scalability.

[0148] Those skilled in the art who wish to increase the growth surface by scaling cultures vertically are encouraged to review Wilson '176, as they will find that many of the embodiments of the present invention can be implemented using the apparatus described in Wilson '176.

[0149] General Description of the Embodiments: As used herein, a growth area refers to the area of ​​a device on which cells reside and is constructed from a gas-permeable material. The gas-permeable material may be any material known to those skilled in the art of cell culture, and is preferably liquid-impermeable and non-porous. The devices and culture methods of the present invention can function without forcing gas through a growth surface constructed from a gas-permeable material. These methods are suitable for static culture.

[0150] Preferred Cell Types: In embodiments of the present invention, if the culture consists of a single cell type, the cells are preferably antigen-presenting cells, more preferably LCL or K562. If the culture consists of a co-culture, it preferably contains effector cells (i.e., cells of interest, target cells) in combination with APCs or feeder cells, with or without beads. Beads are a substitute for APCs or feeder cells. If APCs are present, they are preferably professional antigen-presenting cells, more preferably K562 or LCL types, and more preferably irradiated. If present, and if they are not pancreatic islets, effector cells are preferably derived from peripheral blood or bone marrow, and are more preferably T cells, NK cells, Tregs, TILs, or MILs. If the effector cells are T cells, they are preferably naturally occurring or transduced antigen-specific T cells.

[0151] Preferred Surface Density: In embodiments of the present invention, cells are present on a growth surface comprised of a gas-permeable material at a surface density of preferably 0.5E+06 or less. Based on this disclosure, one skilled in the art will recognize that the surface density can be continuously reduced from 0.5E+06 or less to increase the population expansion rate relative to conventional methods in the field of adoptive cell therapy, and that further reductions are preferred. That is, for example, we have shown that the population expansion rates at surface densities of 0.25E+06, 0.125E+06, and 0.0625E+06 exceed those of conventional methods. Therefore, one skilled in the art should recognize that the surface density need not be limited to the values ​​set forth in our examples, and that possible values ​​are not discrete but are continuous. For example, although we have not provided examples using that specific surface density, we recommend to one of ordinary skill in the art that starting a culture at a surface density of 0.49E+06 will improve the population "expansion fold" compared to starting a culture at a surface density of 0.5E+06. Therefore, those skilled in the art are advised that the surface densities shown in the examples of this invention and in the parent application are to be interpreted analogously and are not limited to the discrete values ​​shown therein.

[0152] Preferred Cell Density: In an embodiment of the present invention, cells reside on a growth surface comprised of a gas-permeable material at a cell density relative to the medium that is preferably 0.5E+06 or less. Given this disclosure, those skilled in the art will recognize that the cell density relative to the medium can be continuously reduced from 0.5E+06 or less, and even more preferably, to reduce the frequency of medium replenishment relative to conventional methods in the field of adoptive cell therapy. Thus, we have shown how the cell density relative to the medium can be reduced from the lower limit of 0.5E+06 cells / ml of conventional methods to reduce the frequency of medium replenishment while maintaining a cell population that can grow at a rate exceeding that of conventional methods. Those skilled in the art should recognize that the surface density need not be limited to the values ​​set forth in our examples, but can take on a continuous range of values ​​rather than discrete values. It is preferable to reduce the cell density relative to the medium to any value below 0.5E+06 that they deem appropriate for the attributes they desire. Thus, while we have described the benefits of reducing the cell density to the same discrete cell density to medium in various examples herein and in the parent application, the invention is not limited to the discrete values ​​shown herein.

[0153] Large Medium Volume to Growth Area Ratio: In this embodiment, the cells reside on a growth surface constructed of a gas-permeable material, which provides benefits from a large medium volume to growth area ratio. Given this disclosure, those skilled in the art will recognize that the medium volume to growth area ratio can be continuously increased to provide numerous benefits when combined with other elements of this invention, such as reduced surface density. Therefore, while these related advantages are described herein and in the parent application using examples with discrete values, this invention is not limited to the discrete values ​​shown. Those of ordinary skill in the art should recognize that the values ​​shown, and combinations of those values, are intended to guide them in interpreting the values ​​continuously to achieve the described benefits. Therefore, this invention is not limited to the discrete numerical values ​​given herein.

[0154] Large Medium Height: In embodiments of the present invention, cells reside on a growth surface constructed of a gas-permeable material, providing benefits by increasing the medium height over conventional methods. Given this disclosure, those skilled in the art will recognize that continuous increases in medium height are possible to provide numerous benefits when combined with other elements of the present invention, such as reduced surface density. Therefore, while these related advantages are described herein and in the parent patents using examples with discrete values, the present invention is not limited to the discrete values ​​shown. Those of ordinary skill in the art should recognize that the values ​​shown, and combinations of those values, are intended to guide them in achieving the described benefits by interpreting those values ​​continuously. Therefore, the present invention is not limited to the discrete numerical values ​​given herein.

[0155] Alternative measurements based on the rate of solute change in the medium instead of cell counting: In embodiments of the present invention, cells reside on a growth surface constructed of a gas-permeable material, providing advantages by allowing the ability to measure how many cells are in culture without the need to count them. Those skilled in the art will recognize that this specification provides examples of how a decrease in glucose concentration can provide a measure of the number of cells in culture. They will also recognize that glucose is only one measurable substrate in the medium utilized by cells, and given this specification, those of ordinary skill in the art will be able to rely on a decrease in the concentration and / or an increase in other substrates in the medium, such as lactate, to indicate cell number. Thus, a novel aspect of embodiments of the present invention is the discovery that substrate measurements in static cultures initiated at any of the following conditions of novel surface density, cell density, medium height, and / or medium volume to growth area ratio are a good way to determine the progress of cell population growth. Therefore, the present invention is not limited to glucose substrate. Therefore, the present invention is not limited to the discrete values ​​given herein.

[0156] Medium Removal Without Cell Loss: In embodiments of the present invention, cells reside at various preferred surface densities on a growth surface comprised of a gas-permeable material, allowing for increased medium height (and / or increased medium volume to growth area ratio) relative to conventional methods. Benefits then arise from lowering the medium height (and / or lowering the medium volume to growth area ratio) relative to conventional methods without cell loss. Given this disclosure, those skilled in the art will recognize that it is possible to improve medium exchange methods (where more equipment does not need to be added to the process) and / or improve cell harvest methods (reducing the medium volume required to harvest cells). While we illustrate these related benefits using examples with discrete values, the present invention is not limited to the discrete values ​​provided herein. Those of ordinary skill in the art should recognize that the provided values ​​and combinations of values ​​are intended to guide them in interpreting the values ​​continuum to achieve the described benefits. For example, the medium can reside at any height, preferably greater than 2.0 cm (e.g., 2.1 cm, 2.5 cm, 6.08 cm, 10.0 cm, etc.). Therefore, the medium removal opening of the medium removal tube, preferably at any height 0.2 cm or more above the growth surface, is preferably at a height of 0.2 cm or higher, as long as it is located below the medium level when removing medium without cell loss, thereby reducing cell separation from the medium and / or allowing the user to avoid having to transfer cells to a separate device when changing medium. The invention is not limited to the discrete numerical values ​​given herein.

[0157] Gas-Permeable Culture Devices: In the embodiments of this invention, we describe discoveries, including improvements to the methods of Wilson '717 and Wilson '176, such as the ability to initiate cultures at lower surface densities than previously known, minimal manipulation for providing cytokines, and media feeding strategies that increase cell population growth rates and reduce production times using novel cell culture devices relative to conventional methods. Furthermore, device manufacturers and providers should recognize that providing gas-permeable devices, including those disclosed in Wilson '717 and Wilson '176, should include the dissemination of the novel methods of this invention and / or the parent patents to users of such devices via instructions, experimental protocols, etc., regardless of form (e.g., paper, electronic media, websites, etc.). Accordingly, the scope of this invention includes the provision of instructions and / or dissemination of the methods of this invention by manufacturers and / or providers of gas-permeable devices.

[0158] The invention being thus described, it will be apparent to one of ordinary skill in the art having the benefit of this specification that the same may be modified in many ways. Such modifications are not to be considered as departing from the spirit and scope of the invention. Such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims and their equivalents.

[0159] Each of the patent applications, patents, and articles cited in this application, and the documents or references cited in each of such patent applications, patents, and articles (including any issued or pending patents; the "Application Citations"), pending U.S. Patent Application Nos. 2005 / 0106717A1 and 2008 / 0227176A1, and each of the PCT and foreign patent applications and patents corresponding to and / or claiming priority from any of these patent applications and patents, and each of the documents cited or referenced in each of these Application Citations, are hereby expressly incorporated by reference.

[0160] The above-mentioned documents are limited by incorporation by reference so as not to incorporate subject matter that is inconsistent with that expressly disclosed herein. The above-mentioned documents are limited by incorporation by reference so that the claims contained therein are not incorporated by reference herein. The above-mentioned documents are limited by incorporation by reference so that definitions provided in those documents are not incorporated herein unless expressly included.

[0161] For purposes of construing a claim to this invention, the provisions of 35 U.S.C. § 112, paragraph 6, should not be applied unless the claim recites the express terms "means for" or "step for."

[0162] Those skilled in the art will recognize that many modifications can be made to this disclosure without departing from the spirit of the invention as set forth herein. Accordingly, there is no intention to limit the breadth of the embodiments and examples described herein. Rather, the scope of the invention should be construed by the appended claims and their equivalents.

Claims

1. A cell culture and cell recovery device for static cell culture, comprising: a cell culture compartment bounded by a growth surface and an upper limit opposite the growth surface; a medium removal tube including a medium removal opening spaced from the growth surface; a cell removal tube in contact with the growth surface; the growth surface comprises a liquid-impermeable and gas-permeable material, the bottom of the gas-permeable material being in contact with a growth surface support, the growth surface support maintaining the gas-permeable material in a horizontal position during culture, and the growth surface support allowing atmospheric gas to contact the bottom surface of the gas-permeable material without forcing it into contact; the cell removal tube includes a cell removal opening disposed along the lower end of the device, such that when the device is oriented for cell collection, the growth surface is not horizontal and the cell removal tube serves to extract medium and cells from a lower portion of the device; An apparatus wherein, when the apparatus is oriented for static cell culture, the growth surface is in a horizontal position and the distance from the growth surface to the medium removal opening is less than half the distance from the growth surface to the upper limit.

2. 10. The device of claim 1, wherein the distance from the growth surface to the medium removal opening is at least 0.2 cm when the device is in the static cell culture position.

3. 10. The device of claim 1, wherein the distance from the growth surface to the medium removal opening does not exceed 2.0 cm.

4. A method for cell culture using the device according to any one of claims 1 to 3, wherein the device contains cells and a culture medium, the device is located in a location with an atmospheric gas suitable for cell culture, and is oriented for static cell culture.

5. The method of claim 4, wherein the cells comprise T cells.

6. 6. The method of claim 4 or 5, wherein the gas permeable material comprises silicone.

7. The method according to any one of claims 4 to 6, wherein the device comprises two or more medium removal openings.

8. The method according to any one of claims 4 to 7, wherein the device is transparent and can be visually evaluated.

9. The method according to any one of claims 4 to 8, wherein the device is rigid.

10. The method according to any one of claims 4 to 6, wherein the cell removal tube extends to a position higher than the upper limit.

11. 11. The method of any one of claims 4 to 10, wherein the atmospheric gas suitable for cell culture contacts the growth surface by random movement without the need for a pump or other mechanism for transferring gas to or from the growth surface.

12. The method of claim 4, wherein the cells are pancreatic islets.

13. 5. The method of claim 4, wherein the highest medium level is parallel to the growth surface.

14. 10. The device of claim 1, wherein the gas permeable material is non-porous.

15. The method according to any one of claims 4 to 13, wherein the medium withdrawal tube is a telescopic tube.

16. 16. The method of claim 15, wherein the cell removal tube is a telescoping tube.

17. The device according to any one of claims 1 to 3, wherein the gas permeable material comprises silicone.

18. 4. The device according to any one of claims 1 to 3, comprising two or more medium removal openings.

19. The device according to any one of claims 1 to 3, which is transparent and can be visually evaluated.

20. The device according to any one of claims 1 to 3, which is rigid.

21. The device according to any one of claims 1 to 3, wherein the cell removal tube extends to a position higher than the upper limit.

22. The device according to any one of claims 1 to 3, wherein the medium withdrawal tube is a telescopic tube.

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